Loss of Bim results in abnormal accumulation of mature CD4-CD8-CD44-CD25- thymocytes

Abstract

The process of thymopoiesis is tightly regulated by a series of selection events which ensure that only functional T-lymphocytes directed against foreign antigens are exported into the periphery. The adaptive immune response largely depends on the regulation of thymocyte development, and thymocytes which fail selection in the thymus are removed by apoptosis. However, the roles of specific apoptotic proteins in early T-lymphocyte development are poorly understood. Here, we report a novel function for Bim in thymocyte development. There is an accumulation of thymocytes in Bim(-/-) mice that lack expression of CD4, CD8, CD44, and CD25 but express CD3 and TCRbeta. Further, the CD4(-)CD8(-)CD25(-)CD44(-)CD3(+)TCRbeta(+) thymocytes are smaller and do not proliferate. These data suggest that these thymocytes are mature DN thymocytes that may have down-regulated the expression of CD4 and CD8. The DN thymocyte phenotype in Bim(-/-) mice is unaffected by the additional loss of Bak or Bax and is similar to the thymic phenotype in mice lacking both Bak and Bax. These data demonstrate that Bim functions to ensure the proper homeostasis of mature thymocytes during selection and thymic export.

Fig. 1

Bim^-/- mice display an altered DN thymocyte population. Single cell and analyzed by flow cytometry. Single cell suspensions of thymocytes from Wt (n=19), Bim^-/- (n=15), Bak^-/-Bax^-/- (n=2), Bak^-/-Bim^-/- (n=6), and Bax^-/-Bim^-/- (n=7) were stained with antibodies for CD4, CD8, CD11b, CD19, CD25, CD44, CD45, and Gr-1 and analyzed for the DN4 stage (CD45⁺CD4^-CD8-CD44^-CD25^-CD11b^-CD19^-Gr-1^-) using flow cytometry. Values represent the mean ± SEM, which were compared by Student’s t-test. * indicates p < 0.05 as compared to Wt. Shown are representative (A) dot plots of DN Wt and Bim^-/- thymocytes and quantitative analysis of the (B) total numbers of DN thymocytes.

Fig. 2

DN4 thymocytes lacking Bim, Bak plus Bax, or Bak plus Bim, or Bax plus Bim are more mature. (A) DN4 thymocytes from Bim^-/- mice have a lower forward light scatter than Wt DN4 thymocytes. Single cell suspensions of thymocytes were stained with antibodies for CD4, CD8, CD11b, CD19, CD44, CD25, and Gr-1 and analyzed by flow cytometry. Shown is a representative histogram of the mean forward light scatter of Wt (black) and Bim^-/- (filled grey) DN4 (CD45⁺CD4^-CD8^-CD44^-CD25^-CD11b^-CD19^-Gr-1^-) thymocytes. (B) Quantitative analysis of DN4 forward light scatter. Wt (n=17), Bim^-/- (n=23), Bak^-/-Bax^-/- (n=2), Bak^-/-Bim^-/- (n=6), and Bax^-/-Bim^-/- (n=7) DN4 thymocytes were analyzed as above for mean forward light scatter. Values represent the mean ± SEM, which were compared by Student’s t-test. * indicates p < 0.04 as compared to Wt. (C) Bim^-/- DN4 thymocytes express higher levels of extracellular TCRβ than Wt DN4 thymocytes. Single cell suspensions of thymocytes were stained with antibodies for CD4, CD8, CD11b, CD19, CD44, CD25, Gr-1, and TCRβ and analyzed by flow cytometry. Shown is a representative histogram of the mean fluorescent intensity of TCRβ expression in Wt (black) and Bim^-/- (filled grey) DN4 (CD45⁺CD4^-CD8^-CD44^-CD25^-CD11b^-CD19^-Gr-1^-) thymocytes. (D) Quantitative analysis of DN4 extracellular TCRβ expression. Wt (n=19), Bim^-/- (n=15), Bak^-/-Bim^-/- (n=6), and Bax^-/-Bim^-/- (n=7) DN4 thymocytes were analyzed as described in the Materials and Methods for TCRβ extracellular expression. Values represent the mean ± SEM, which were compared by Student’s t-test. * indicates p < 0.01 as compared to Wt. (E) Bim^-/- and Wt DN4 thymocytes have similar expression of intracellular TCRβ. Single cell suspensions of thymocytes were stained with antibodies for CD4, CD8, CD11b, CD19, CD44, CD25, Gr-1, and TCRβ and analyzed by flow cytometry. Shown is a representative histogram of the mean fluorescent intensity of TCRβ expression in Wt (black) and Bim^-/- (filled grey) DN4 (CD45⁺CD4^-CD8^-CD44^-CD25^-CD11b^-CD19^-Gr-1^-) thymocytes. (F) Quantitative analysis of DN4 intracellular TCRβ expression. Wt (n=11), Bim^-/- (n=3), Bak^-/-Bim^-/- (n=4), and Bax^-/-Bim^-/- (n=5) DN4 thymocytes were analyzed as described in the Materials and Methods for TCRβ intracellular expression. N.D. = not determined. Values represent the mean ± SEM, which were compared by Student’s t-test.

Fig 3

Bim^-/- mature DN and TN thymocytes display reduced proliferation. (A) Fewer DN Bim^-/- thymocytes incorporate BrdU. Thymocytes were isolated from BrdU-injected mice as described in Materials and Methods. Shown are representative dot plots of anti-BrdU staining in the mature DN and TN thymic populations from Wt (n=12) and Bim^-/- (n=12) mice. (B) Quantitative analysis of percent thymic proliferation. Values represent the mean ± SEM, which were compared by Student’s t-test. * indicates p < 0.0001 as compared to Wt.