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. Author manuscript; available in PMC: 2013 Dec 2.

Published in final edited form as: J Immunol. 2009 Mar 15;182(6):10.4049/jimmunol.0803121. doi: 10.4049/jimmunol.0803121

Functional impairment of 125Tg immature B cells is highly reversible. 125Tg immature B cells were cultured (Fig. 2) with continuous Ag (days 1–2) or Ag was withdrawn from the culture (Ag added day 1, removed day 2). IgM down-regulation, calcium mobilization, and proliferation were assessed. A, Flow cytometric analysis showing relative IgM surface expression levels, as calculated in Fig. 3A. The average of eight animals ± SD is shown. B and C, Intracellular Ca²⁺ mobilization was measured in cells cultured with 0 ng/ml (solid black line), 500 ng/ml (dashed gray line), or 5 × 10⁴ ng/ml human insulin (dashed black line) for 2 days (continuous insulin presence, B) or 1 day, followed by Ag removal and 1 day of additional culture in the absence of insulin (insulin withdrawal, C) and stimulated with human insulin. Data are representative of four animals. D, [³H]thymidine incorporation (cpm) was measured. Five hundred nanograms of human insulin per milliliter was absent for 3 days (□), continuously present for 3 days (), or added for 1 day and then removed for 2 days (striped gray bars); cells were stimulated with anti-CD40 (x-axis) for the final 2-day period. The average of triplicate values ± SD is shown; data are representative of two animals. A two-tailed, two-sample/unequal variance t test was used to calculate the significance for all bar graphs, *, p < 0.001 and **, p < 0.0007.

Inline graphic — Functional impairment of 125Tg immature B cells is highly reversible. 125Tg immature B cells were cultured (Fig. 2) with continuous Ag (days 1–2) or Ag was withdrawn from the culture (Ag added day 1, removed day 2). IgM down-regulation, calcium mobilization, and proliferation were assessed. A, Flow cytometric analysis showing relative IgM surface expression levels, as calculated in Fig. 3A. The average of eight animals ± SD is shown. B and C, Intracellular Ca²⁺ mobilization was measured in cells cultured with 0 ng/ml (solid black line), 500 ng/ml (dashed gray line), or 5 × 10⁴ ng/ml human insulin (dashed black line) for 2 days (continuous insulin presence, B) or 1 day, followed by Ag removal and 1 day of additional culture in the absence of insulin (insulin withdrawal, C) and stimulated with human insulin. Data are representative of four animals. D, [³H]thymidine incorporation (cpm) was measured. Five hundred nanograms of human insulin per milliliter was absent for 3 days (□), continuously present for 3 days (), or added for 1 day and then removed for 2 days (striped gray bars); cells were stimulated with anti-CD40 (x-axis) for the final 2-day period. The average of triplicate values ± SD is shown; data are representative of two animals. A two-tailed, two-sample/unequal variance t test was used to calculate the significance for all bar graphs, *, p < 0.001 and **, p < 0.0007.