Figure 7
Figure 7. Effect of anti-CD38 mAb on antigen-induced T-cell activation and cytokine production. (A) IFN-γ production by CH7C17 T cells incubated overnight with HA peptide–pulsed (+) or unpulsed (−) HOM-2 B cells. CH7C17 T cells were preincubated for 15 minutes at 37°C with the indicated anti-CD38 mAb or with reagents at the concentrations indicated in “Results.” Representative experiment out of 3 independent experiments performed in triplicates (mean ± SEM). Statistical analysis: unpaired t test; n.s., not significant. (B) IL-2, IFN-γ, and IL-10 production (and 7 additional cytokines) was assayed simultaneously by a multiplex system (“Methods”) in supernatants of CH7C17 T cells stimulated as in panel A. Representative experiment out of 5 independent experiment is shown. (C) IL-2 production by Jurkat J77 T cells incubated overnight with SEE-pulsed (+) or unpulsed (−) Raji B cells. Jurkat J77 T cells were preincubated for 15 minutes at 37°C with the indicated anti-CD38 mAb or with reagents at the concentrations indicated in “Results.” Representative experiment out of 3 independent experiments performed in triplicates (mean ± SEM). Statistical analysis: unpaired t test; n.s., not significant. (D) After removal of supernatants in 5 experiments as in panel B, cells were tested for CD69 (■), CD25 (▨), or CD38 (□) expression by FACS. The data represent the fold increase over the geometric mean values obtained in CH7C17 T cells stimulated with non–HA-pulsed HOM2 cells in the absence of IB6 (1-fold value), and are the mean plus SE of 5 independent experiments. (E) Western blot analysis with the anti–phospho-PKCθ (Thr538), followed by anti-PKCθ for protein-loading control of cell lysates from CH7C17 T cells incubated with unpulsed (−HA) or HA-pulsed (+HA) HOM-2 B cells. CH7C17 T cells were either preincubated with vehicle (−IB6) or preincubated for 30 minutes at 37°C with the anti-CD38 mAb IB6 (+IB6), then washed to eliminate unbound IB6, mixed with HOM-2 B cells, and incubated at 37°C for the indicated periods of time. The image is representative of 3 independent experiments. (F) The level of PKCθ phosphorylation at Thr538 (y-axis) was plotted against time (minutes). ■ indicates CH7C17 T cells incubated with unpulsed HOM-2 cells for the indicated periods of time; □, CH7C17 T cells preincubated with anti-CD38 mAb IB6 and then with unpulsed HOM-2 cells; ●, CH7C17 cells incubated with HA-pulsed HOM-2 cells; and ○, CH7C17 cells preincubated with IB6 and then incubated with HA-pulsed HOM-2 cells. (G) CH7C17 T cells were preincubated with different concentrations of IB6 or vehicle as indicated. After 30 minutes at 37°C, cells were washed and incubated with either nonpulsed (−HA) or HA-pulsed (+HA) HOM-2 B cells for 20 minutes. Western blotting of cell lysates were probed with anti–phospho-PKCθ (Thr538), followed by anti-PKCθ total for protein-loading control. (H) The level of phosphorylation of PKCθ at Thr538 (y-axis) in CH7C17 T cells interacting with either unpulsed (□) or HA-pulsed (■) APCs was plotted against the concentration of IB6 mAb used (x-axis).

Effect of anti-CD38 mAb on antigen-induced T-cell activation and cytokine production. (A) IFN-γ production by CH7C17 T cells incubated overnight with HA peptide–pulsed (+) or unpulsed (−) HOM-2 B cells. CH7C17 T cells were preincubated for 15 minutes at 37°C with the indicated anti-CD38 mAb or with reagents at the concentrations indicated in “Results.” Representative experiment out of 3 independent experiments performed in triplicates (mean ± SEM). Statistical analysis: unpaired t test; n.s., not significant. (B) IL-2, IFN-γ, and IL-10 production (and 7 additional cytokines) was assayed simultaneously by a multiplex system (“Methods”) in supernatants of CH7C17 T cells stimulated as in panel A. Representative experiment out of 5 independent experiment is shown. (C) IL-2 production by Jurkat J77 T cells incubated overnight with SEE-pulsed (+) or unpulsed (−) Raji B cells. Jurkat J77 T cells were preincubated for 15 minutes at 37°C with the indicated anti-CD38 mAb or with reagents at the concentrations indicated in “Results.” Representative experiment out of 3 independent experiments performed in triplicates (mean ± SEM). Statistical analysis: unpaired t test; n.s., not significant. (D) After removal of supernatants in 5 experiments as in panel B, cells were tested for CD69 (■), CD25 (▨), or CD38 (□) expression by FACS. The data represent the fold increase over the geometric mean values obtained in CH7C17 T cells stimulated with non–HA-pulsed HOM2 cells in the absence of IB6 (1-fold value), and are the mean plus SE of 5 independent experiments. (E) Western blot analysis with the anti–phospho-PKCθ (Thr538), followed by anti-PKCθ for protein-loading control of cell lysates from CH7C17 T cells incubated with unpulsed (−HA) or HA-pulsed (+HA) HOM-2 B cells. CH7C17 T cells were either preincubated with vehicle (−IB6) or preincubated for 30 minutes at 37°C with the anti-CD38 mAb IB6 (+IB6), then washed to eliminate unbound IB6, mixed with HOM-2 B cells, and incubated at 37°C for the indicated periods of time. The image is representative of 3 independent experiments. (F) The level of PKCθ phosphorylation at Thr538 (y-axis) was plotted against time (minutes). ■ indicates CH7C17 T cells incubated with unpulsed HOM-2 cells for the indicated periods of time; □, CH7C17 T cells preincubated with anti-CD38 mAb IB6 and then with unpulsed HOM-2 cells; ●, CH7C17 cells incubated with HA-pulsed HOM-2 cells; and ○, CH7C17 cells preincubated with IB6 and then incubated with HA-pulsed HOM-2 cells. (G) CH7C17 T cells were preincubated with different concentrations of IB6 or vehicle as indicated. After 30 minutes at 37°C, cells were washed and incubated with either nonpulsed (−HA) or HA-pulsed (+HA) HOM-2 B cells for 20 minutes. Western blotting of cell lysates were probed with anti–phospho-PKCθ (Thr538), followed by anti-PKCθ total for protein-loading control. (H) The level of phosphorylation of PKCθ at Thr538 (y-axis) in CH7C17 T cells interacting with either unpulsed (□) or HA-pulsed (■) APCs was plotted against the concentration of IB6 mAb used (x-axis).

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