Figure 1.
Treg cell numbers are reduced in IκBα-SR Tg mice.
Single-cell suspensions from thymus and spleen of wild-type (WT) and IκBα-“SuperRepressor” transgenic (IkB-SR Tg) mice were stained with CD4, CD8, CD25, and Foxp3 antibodies and analyzed by flow cytometry. (A, C) Representative dot plots from one of three independent experiments are shown. Expression profiles of CD4 versus CD8 (upper panels) and CD25 versus intracellular Foxp3 (gated on CD4 SP cells, lower panels) of thymocytes (A) and splenocytes (C) of WT and IkB-SR Tg mice. Numbers in the quadrants indicate the percentages of cells. (B, D) Percentages of CD25+Foxp3+ Treg cells relative to CD4SP cells, as well as absolute numbers of Treg cells in the thymus (B) and spleen (D) of individual WT and IkB-SR Tg mice are displayed. Each symbol represents an individual mouse. Data were compiled from three independent experiments. In each experiment, organs from three mice per genotype were analyzed separately. Horizontal bars represent the mean. Mann-Whitney U-test was used for statistical analyses. *, p≤0.05; ***, p≤0.001; ns, not significant.
Figure 2.
IL-2 can only partially rescue Treg cell numbers in the thymus of IκBα-SR Tg mice.
(A) Thymocytes from wild-type (WT) and IκBα-“SuperRepressor” transgenic (IkB-SR Tg) mice were either left untreated (−) or stimulated (+) with PMA and ionomycin for 24 h. The IL-2 concentrations in the supernatants were determined by ELISA. IL-2 secretion from unstimulated cells was below the detection limit. Mean values and SD were calculated from triplicates. Student's t test was used for statistical analyses. *, p≤0.05. (B, C, D) Wild-type and IkB-SR Tg mice were injected either with IL-2 or PBS every 8 h for 2 days and thymi were analyzed by flow cytometry. (B) Representative dot plots from one of three independent experiments are shown. Numbers in the quadrants represent the percentages of cells among CD4SP thymocytes from untreated (PBS) and IL-2 treated mice. (C) Percentages of CD25+Foxp3− cytokine-responsive Treg precursor cells among CD4SP thymocytes, as well as their absolute numbers in the thymus. (D) Percentages of CD25+Foxp3+ mature Treg cells among CD4SP thymocytes, as well as their absolute numbers. (B, C, D) Data were compiled from two independent experiments with three mice per genotype and treatment in each experiment. Each symbol represents an individual mouse. Horizontal bars represent the mean. Mann-Whitney U-test was used for statistical analyses. *, p≤0.05; ns, not significant.
Figure 3.
Treg cell development is intrinsically impaired in thymocytes overexpressing an IκBα-“SuperRepressor”.
Mixed bone marrow chimeras were generated by either injecting equal numbers of CD45.1+C57BL/6 wild-type and CD45.2+IkB-SR Tg bone marrow cells (mixed chimera, n = 5) or just CD45.1+C57BL/6 wild-type bone marrow cells (WT chimera, n = 3) into lethally irradiated Rag1−/− recipient mice. Ten weeks later, Treg populations were examined in thymus, spleen and peripheral blood by flow cytometry. (A) Percentages of Treg cells in respect to CD45.1+ or CD45.2+ CD4SP cells as indicated in organs of mixed chimeras. Connected symbols indicate values from the same mouse. (B, C). Contributions of wild-type (CD45.1+) and IkB-SR Tg derived (CD45.2+) cells to the total Treg cell populations in thymus, spleen, and peripheral blood of mixed chimeras. Depicted are the mean values of percentages (B) as well as absolute cell numbers + SD (C) of contributing cells in the Treg population. Data are representative of two independent experiments with similar numbers of mice in each experimental group. Mann-Whitney U-test was used for statistical analyses. **, p≤0.01; ns, not significant.
Figure 4.
Pharmacological NF-κB inhibition impairs thymic Treg cell development.
Wild-type mice either received the IKKβ-inhibitor AS602868 (30 mg/kg) or vehicle twice a day for 7 days and thymus and spleen were analyzed by flow cytometry. Representative dot plots from one of three independent experiments are shown (upper panel). Numbers in the quadrants represent the percentages of CD25+Foxp3+ cells among CD4SP cells in thymus (A) and spleen (B) from untreated (Vehicle) and treated mice (AS602868). Percentages of CD25+Foxp3+ Treg cells among CD4SP cells of individual mice are displayed (lower panel). Data are representative of three independent experiments with equal numbers of mice in each experimental group. Each symbol represents an individual mouse. Horizontal bars represent the mean. Mann-Whitney U-test was used for statistical analyses. *, p≤0.05; **, p≤0.01; ns, not significant.