Interleukin-7 regulates Bim proapoptotic activity in peripheral T-cell survival

Abstract

Interleukin-7 (IL-7) is critical for T-cell development and peripheral T-cell homeostasis. The survival of pro-T cells and mature T cells requires IL-7. The survival function of IL-7 is accomplished partly through induction of the antiapoptotic protein Bcl-2 and inhibition of proapoptotic proteins Bax and Bad. We show here that the proapoptotic protein Bim, a BH3-only protein belonging to the Bcl-2 family, also plays a role in peripheral T-cell survival. Deletion of Bim partially protected an IL-7-dependent T-cell line and peripheral T cells, especially cells with an effector memory phenotype, from IL-7 deprivation. However, T-cell development in the thymus was not restored in IL-7(-/-) Rag2(-/-) mice reconstituted with Bim(-/-) bone marrow. IL-7 withdrawal altered neither the intracellular location of Bim, which was constitutively mitochondrial, nor its association with Bcl-2; however, a reduction in its association with the prosurvival protein Mcl-1 was observed. IL-7 withdrawal did not increase Bim mRNA or protein expression but did induce changes in the isoelectric point of Bim(EL) and its reactivity with an antiphosphoserine antibody. Our findings suggest that the maintenance of peripheral T cells by IL-7 occurs partly through inhibition of Bim activity at the posttranslational level.

Figures

FIG. 1.

Bim induces the death of IL-7-dependent D1 cells. (A) D1 cells were infected with retroviruses expressing either GFP alone (pMIG) or isoforms of Bim and were cultured at 37°C for 16 h in the presence of 50 ng/ml of IL-7. Apoptotic cells were determined by annexin V/7-AAD staining. The proportion of apoptotic cells was calculated by annexin V positivity and was expressed as a percentage of vector (pMIG)-infected cells (GFP-positive cells). Values are means ± standard deviations from three experiments. (B) D1 cells were infected with retroviruses carrying either a scrambled control siRNA or a siRNA specific for Bim (shBim18). GFP-positive cells were sorted after 24 h of infection and were cultured with 50 ng/ml of IL-7. Total-cell extracts were subjected to immunoblotting with an antibody against Bim. (C) D1 cells or D1 cells in which Bim had been knocked down by siRNA were deprived of IL-7 for 24 h or 48 h, and cell death was quantitated by annexin V/7-AAD staining. Values are means ± standard deviations for three experiments (*, P ≤ 0.001; **, P = 0.002).

FIG. 2.

Bim−/− T cells are more resistant to IL-7 deprivation than WT T cells in vitro. CD4- or CD8-positive T cells were isolated from the LNs of individual Bim−/− or WT mice by negative selection with magnetic beads. The purified cells were cultured either with IL-7 (2 ng/ml) or without IL-7 for the indicated time points. Cell viability was measured by annexin V/7-AAD staining. Results are means ± standard deviations for six individual mice in each group. P values for comparison of Bim−/− versus WT CD4 cells are as follows: *, 0.001; **, ≤0.0001; ***, ≤0.0007. P values for comparison of Bim−/− versus WT CD8 cells are as follows: *, 0.002, **, ≤0.0001; ***, ≤0.0001).

FIG. 3.

Loss of Bim improves T-cell survival but not proliferation in IL-7-deficient hosts. (A) T cells were isolated from LNs of Bim−/− or WT mice, labeled with CFSE, and then transferred intravenously into irradiated IL-7−/− mice. Six days later, spleen cells were stained for CD4 and CD8. Donor T cells were analyzed for CFSE-positive cells by gating on CD4 and CD8. Numbers of CFSE-positive cells in different groups were determined for each spleen. Data were analyzed using CellQuest software (BD Biosciences). Means ± standard deviations for 2 or 3 mice from two independent experiments are shown (*, P = 0.008; **, P ≤ 0.001). (B) CFSE-positive cells were recovered from WT recipient spleens after 6 days of adoptive transfer of T cells. (C) Six days after transfer, T cells were recovered from the spleens of IL-7−/− or WT recipient mice. Donor T-cell homeostatic proliferation profiles were analyzed for CFSE intensity by gating on CD4 or CD8 using flow cytometry. Numbers above the peaks indicate the number of cell divisions. Vertical lines represent the gate used for calculating the percentage of cells that had divided. (D) T cells from Bim−/− or WT mice were transferred to irradiated Rag2−/− or IL-7−/− Rag2−/− recipients. Eight weeks later, spleens were harvested, and CD4 or CD8 T-cell recovery was determined by staining with anti-CD4 and anti-CD8.

FIG. 4.

Lack of Bim does not relieve the block in T-cell development caused by the absence of IL-7. Bone marrow from Bim−/− or WT mice was used to reconstitute irradiated Rag2−/− or IL-7−/− Rag2−/− recipients. Eight weeks later, thymuses were harvested from recipients. (A) Thymic cellularity for individual recipients. Means ± standard deviations for 2 to 3 mice from two independent experiments are shown (*, P = 0.009; *, P = 0.001). (B) Thymocytes were stained for CD4 and CD8 (top) or TCRαβ and TCRδγ (bottom). Cell percentages for each quadrant are shown in the upper right corner. Values represent data for five mice from two individual experiments.

FIG. 5.

IL-7 does not alter Bim synthesis or mitochondrial translocation. Cells were placed in culture for 12 h with or without IL-7. (A) IL-3 was withdrawn from FL5.12A cells for 12 h. Total-cell extracts were subjected to immunoblotting with an antibody against Bim. (B) Total-cell extracts from D1 or LN cells were resolved by SDS-PAGE and subjected to immunoblotting with anti-Bim. (C) Primary T cells were isolated from spleens and LNs of 5 WT mice by negative selection with magnetic beads and were placed in culture for 10 h with or without IL-7. D1 or primary T cells were fractionated into cytoplasmic and mitochondrial fractions, and Bim protein levels were visualized by probing blots with an antibody specific for Bim. Cytochrome c oxidase and actin are shown as markers for mitochondrial and cytosolic proteins, respectively. (D) Cell extracts were prepared from D1 or primary T cells, immunoprecipitated (IP) with anti-Bcl-2, and blotted with anti-Bim or anti-Bcl-2. (E) Total proteins extracted from D1 or primary T cells were subjected to immunoprecipitation with anti-Bim, followed by blotting with anti-Bcl-2, anti-Mcl-1, and anti-Bim. The relative association of Bcl-2 or Mcl-1 with BimEL was quantified using an ImageJ program by measuring densitometry after normalization against BimEL protein.

FIG. 6.

IL-7 regulates Bim at the posttranslational level. (A) D1 cells were cultured in the present or absence of IL-7. Total-cell extracts were subjected to 2D gel electrophoresis and immunoblotting with anti-Bim. (B) Cell extracts were prepared from D1 cells cultured with or without IL-7, immunoprecipitated with anti-Bim, and treated with CIAP, followed by blotting with anti-phosphoserine 4A4 and anti-Bim. (C) D1 cells were infected with retroviruses expressing either GFP alone (pMIG), WT BimEL, or mutants of BimEL. The infected cells were cultured at 37°C for 16 h in the present of 50 ng/ml of IL-7. Apoptotic cells were determined by annexin V/7-AAD staining. The proportion of apoptotic cells was calculated as the percentage of vector (pMIG)-infected cells (GFP-positive cells) that were positive for annexin V. Values are means ± standard deviations for three experiments (*, P = 0.01; **, P = 0.042; ***, P ≤ 0.0098).