Figure 3.

OX40 signaling induces stimulation of metabolic activity and cytokine release in ALL cells.

PBMC of ALL patients were either cultured alone, on immobilized M-OX17 monoclonal antibody or human IgG isotype control (10 μg/ml).

(A) Metabolic activity of ALL cells was determined by CellTiter Glo assay after 24 hours. Results of OX40-positive (n=10) and OX40-negative samples (n=3) are shown. For normalization, results obtained with untreated ALL cells were set to 1. Horizontal bars represent mean.

(B) Cytokine levels of IL-6 (after 24 hours), TNF, and IL-8 (both after 6 hours) in culture supernatants were determined by ELISA. Results obtained with OX40-positive (n=14, upper panel) and OX40-negative ALL samples (n=3, lower panel) are shown. Horizontal bars represent mean.

(C) Fold increase (OX40/isotype) in cytokine production (IL-6, TNF, and IL-8) of ALL cells after OX40 stimulation. Each patient is shown as individual bar. Dotted lines represent a 2.5-fold increase in cytokine production.

(D) Quantification of cytokine release by ALL cells after OX40 stimulation. Numbers of patients showing fold increase <2.5 (light gray), ≥2.5-fold increase for one cytokine (gray), two cytokines (dark grey) or three cytokines (black) are displayed.

(E) Analysis of results obtained with 14 OX40-positive patient samples with regard to release of specific cytokine combinations. Positive response (+) was defined as ≥2.5-fold increase of each individual cytokine upon OX40 signaling. The percentage of samples responding with the indicated cytokine pattern is depicted.

*Statistically significant differences, P<.05. ns, not significant.