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. Author manuscript; available in PMC: 2009 Oct 6.
Published in final edited form as: Eur J Immunol. 2009 Mar;39(3):763–775. doi: 10.1002/eji.200838657

Figure 4. Marked increase in intracellular Bcl-2 expression in NKT and NK1.1− T cells that survive after irradiation.

Figure 4

(A) Splenocytes were harvested from untreated C57BL/6 mice or from mice treated with TBI 24 hrs after irradiation, stained for NK1.1 vs TCRαβ, fixed and permeabilized, and stained for intracellular Bcl-2. Dead cells were excluded from the analysis by adding the dye EMA to the staining mixture, and gating on EMA− cells. The left column of panels show the representative analyses of light scatter versus Bcl-2 amongst gated NK1.1+ TCRαβ+ T cells and the right column shows the analyses amongst gated NK1.1− TCRαβ+ T cells. Boxes enclosed either Bcl-2lo (left) or Bcl-2hi (right) cells, and percentages in boxes are shown. The mean percentage of Bcl-2hi NKT cells among all NKT cells in the spleen of untreated mice was 10.9 % ± 3.26%, and increased to 18.7% ± 0.75%; p<0.001, 39.3% ± 14.55%; p<0.001, 77.8% ± 16.57%, p<0.0001, 84.9% ± 8.49%; p<0.0001 in mice given 240, 1,000, 2,000 and 3,000 cGy respectively. There are 8 to 10 mice per group. Thresholds for Bcl-2hi cells were set between the two peaks of Bcl-2 staining after one color analysis of gated NKT cells from untreated mice shown in lower left panel (shaded profile). One color profile of staining with irrelevant isotype matched mAb is shown by dashed line. (B, C) The mean absolute number of total or Bcl-2hi NKT cells and NK1.1− T cells in the spleen of untreated mice (UNT) and in the spleen 24 hours after each dose of TBI (1,000, 2,000 and 3,000 cGy) are shown on a logarithmic scale. There are 10 to 20 mice in each group. (D) The percentage of NKT cells in the spleen of human Bcl-2 transgenic (Tg) C57BL/6 mice fails to increase after irradiation. Bcl-2 transgenic mice were analyzed for the percentage of NKT cells among all T cells in untreated mice or after 1,000 cGy of TBI. Left panels show forward scatter versus TCRαβ; right panels show NK1.1 versus TCRαβ on gated TCRαβ+ cells; graph shows changes in mean absolute numbers of NK1.1− T and NKT cells. Untreated and irradiated wild type mice are shown for comparison. The data are representative of three independent experiments.