Journal: The Journal of Experimental Medicine

Article Title: Inherited human OX40 deficiency underlying classic Kaposi sarcoma of childhood

doi: 10.1084/jem.20130592

Figure Lengend Snippet: R65C is a loss-of-function mutation. (A) PHA-activated T cell blasts from healthy controls (C), R65C heterozygous family members (Het), or the patient (P) were incubated with biotinylated recombinant soluble OX40L. Unbound OX40L molecules were washed out, and the levels of cell-bound OX40L were measured by flow cytometry with allophycocyanin-labeled streptavidin (SA-APC). Representative histograms and MFIs (mean fluorescence intensities) of CD3 + CD4 + cells (CD4 + T cells) and CD3 + CD8 + cells (CD8 + T cells) in three independent experiments are shown. (B) PHA-activated T cell blasts from the patient were transduced with bicistronic lentiviral vectors encoding luciferase (Luc), OX40-WT, or OX40-R65C, together with IRES-RFP. Cell surface OX40 levels measured with ACT35 and binding to OX40L measured with biotinylated recombinant soluble OX40L are shown for CD3 + CD4 + RFP + cells. One result representative of two independent experiments is shown. (C) OX40L binding to Jurkat or HEK-293 cells transduced with bicistronic retroviruses with an empty vector (Mock) or encoding OX40-WT or OX40-R65C, together with IRES-GFP, was assessed as in A. The MFIs of OX40L binding for GFP + cells, normalized with respect to those for isotype controls, are shown. The mean of three independent experiments is shown. Error bars indicate the SEM. *, P < 0.05. (D) CFSE-labeled PBMCs from healthy controls (C1–C8) and the patient (P) were incubated for 3 d with 1 ng/ml of plate-bound anti-CD3 antibody and Vero cells infected with retroviruses either with an empty vector (Vero-Mock) or encoding OX40L (Vero-OX40L). Percentages of CFSE-diluted cells in the CD3 + CD4 + population are plotted. One result representative of two independent experiments is shown. (E) CFSE-labeled PBMCs from healthy controls (C1–C8) and the patient (P) were incubated for 3 d with the indicated concentrations of plate-bound anti-CD3 antibody or PHA. Percentages of CFSE-diluted cells in the CD3 + CD4 + population are plotted. One result representative of two independent experiments is shown.

Article Snippet: The final concentrations and the source of recall antigens were as follows: 5 µg/ml tuberculin PPD, 7 Lf/ml TT (Statens Serum Institut), uninfected normal human dermal fibroblast (NHDF) extract, CMV-infected NHDF extract, VZV-infected NHDF extract, uninfected Vero cell extract, HSV-1–infected Vero cell extract, and 2.5–5 µg/ml EBV-infected human B cell extract (EastCoast Bio).

Techniques: Mutagenesis, Incubation, Recombinant, Flow Cytometry, Labeling, Fluorescence, Transduction, Luciferase, Binding Assay, Plasmid Preparation, Infection

Journal: The Journal of Experimental Medicine

Article Title: Inherited human OX40 deficiency underlying classic Kaposi sarcoma of childhood

doi: 10.1084/jem.20130592

Figure Lengend Snippet: Impaired CD4 + T cell recall antigen response. (A and C) PBMCs from eight healthy controls (C1–C8), an R65C heterozygous family member (I.1), and the patient (P) were stimulated with PPD or PHA for 5 d. IFN-γ (A) or IL-10 (C) levels in the supernatant were measured by ELISA. The dotted lines indicate the limit of detection. One result representative of three independent experiments is shown. BCG+, BCG vaccinated; BCG−, no prior BCG vaccination; NS, nonstimulated. (B and D) IFN-γ (B) or IL-10 (D) production in response to various recall antigens was assessed as in A and C. Except for TT, which was provided as purified protein, the recall antigens were provided as virus-infected crude cell lysate. A lysate of uninfected cells tested in the same experiment did not trigger IFN-γ production (not depicted). The dotted lines indicate the limit of detection. One result representative of three independent experiments is shown. (E) CFSE-labeled PBMCs were incubated with the indicated recall antigens for 6 d. T cell proliferation was assessed by determining the proportion of cells with CFSE levels lower than the undivided peak. Results are shown for CD4 + T cells (CD3 + CD4 + ). One result representative of three independent experiments is shown. (F) PBMCs from two healthy controls were incubated with 1 ng/ml of plate-bound anti-CD3 and Vero cells infected with retroviruses with an empty vector (Vero-Mock) or encoding OX40L (Vero-OX40L) for 3 d, or PPD or TT for 6 d. PBS, isotype antibody, or anti-OX40L antibody was added every other day to a concentration of 1 µg/ml. IFN-γ levels in the culture supernatant were measured by ELISA. Means and SEM from two experiments for one of the healthy controls are shown. **, P < 0.01; ns, not significant.

Article Snippet: The final concentrations and the source of recall antigens were as follows: 5 µg/ml tuberculin PPD, 7 Lf/ml TT (Statens Serum Institut), uninfected normal human dermal fibroblast (NHDF) extract, CMV-infected NHDF extract, VZV-infected NHDF extract, uninfected Vero cell extract, HSV-1–infected Vero cell extract, and 2.5–5 µg/ml EBV-infected human B cell extract (EastCoast Bio).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Infection, Labeling, Incubation, Plasmid Preparation, Concentration Assay