T cell survival and function requires the c-Abl tyrosine kinase

Abstract

C-Abl (Abl) regulates multiple cellular processes, including proliferation, survival, shape determination and motility, and participates in cellular responses to genotoxic and oxidative stress stimuli. Mice lacking Abl exhibit retarded growth, osteoporosis and defects in the immune system resulting in lymphopoenia and susceptibility to infections, leading to early death. To define the role of Abl in the regulation of adult T cells we ablated Abl exclusively in T cells by generating mice with floxed abl alleles and expressing an Lck-Cre transgene (Abl-T(-/-)). These mice exhibited thymic atrophy and abnormally reduced T cell numbers in the periphery. The thymic atrophy was caused by increased susceptibility of thymocytes to cell death. Importantly, Abl deficient T cells displayed abnormally reduced response to mitogenic stimulation in vitro. Consequently, Abl-T(-/-) mice exhibited impaired ability to reject syngeneic tumor, to induce T-mediated tumor cell killing, and to generate anti-tumor antibodies. These results demonstrate a cell-autonomous role for Abl in T cell function and survival.

Figure 1

Ablation of Abl in T cells causes thymic atrophy and a reduction in the numbers of mature T cells in peripheral lymphoid organs. (A) Extracts from freshly isolated thymi or spleens from control or Abl-T^−/− mice were subjected to Western blot analysis. Activated T cells were generated by stimulating spleen cells with ConA (72 hrs) followed by stimulation with IL-2 (48 hrs). The levels of the Abl protein were determined by immunoblotting with antibody to mouse Abl, followed by probing with antibody to actin (loading control). (B) Freshly isolated thymocytes derived from control (black solid line), or Abl-T^−/− (broken black line) mice were fixed and stained with antibodies to mouse Abl followed by FITC-conjugated goat anti-mouse secondary antibody. The levels of Abl were monitored by flow cytometric analysis. Background staining with secondary antibody alone is shown by the grey line. (C) Representative images of thymi and spleens from 4 week old control and Abl-T^−/− mice. (D and E) Total numbers of viable cells in thymi and spleens from control or Abl-T^−/− mice were determined by counting trypan excluding blue cells in a hemocytometer. The number of cells in the wt mice was considered as 100%. The data represent 8 mice in each group analyzed in 8 independent experiments. (F) The numbers of viable B cells and T cells in freshly isolated spleens, (G) lymph nodes (LN), and peripheral blood (H) from control or Abl-T^−/− mice were determined by cell counting and immunofluorescent staining with antibodies for T (Thy1.2), B (B220) and T cell sub-population CD4/CD8 specific markers followed by FACS analysis. Data represent 6 (spleen and LN) or 10 mice peripheral blood cells(PBC) from each group. Statistics: student t-test (two tailed) = *p < 0.05, **p < 0.01, ***p < 0.001, ^§p < 0.0001.

Figure 2

Loss of Abl slightly increases the relative number of DN cells with no significant effect on the other three thymic subpopulations, and does not affect proliferation of thymocytes. (A) Thymocytes from control or Abl-T^−/− mice were examined for the four major thymic sub-populations by FACS after staining with antibodies to Thy1.2 (FITC), CD4 (PE) and CD8 (APC). Representative data are shown in dot blots (right) and a summary of at least 6 mice (4–6 weeks) from each genotype is shown. Similar results were obtained with older mice (8–10 weeks data not shown). Thymocytes from control or Abl-T^−/− mice were examined for the double negative thymic sub-population by flow after staining with antibodies to CD4, CD8, CD25 and CD44. Analysis was performed on CD8⁻ CD4⁻ DN population average of 5 mice in each group. (B) BrdU incorporation assay was performed on freshly isolated thymocytes. Cells were pulsed with BrdU (20 μM) for 1 hr, then fixed and stained with FITC-conjugated anti-BrdU antibody (Dako Cytomation) and were subjected to flow cytometric analysis. BrdU positive cells were calculated among the gated diploid population. Representative results are shown in (B and C), by dot plot (BrdU on the Y axis and DNA content on the X axis) or histogram (control—black solid line, Abl-T^−/− broken black line), respectively. A summary of data from 5 mice of each genotype is shown in (D).

Figure 3

Abl deficient thymocytes show increased propensity for spontaneous cell death. Freshly isolated thymi from 17–20 days old control or Abl-T^−/− mice were fixed, paraffin embedded and slides were stained for active caspase-3 antibodies (Cell Signaling) followed by F(ab)2 fragments of Cy5-conjugated goat anti-rabbit IgG antibodies (Jackson laboratories) for the detection of apoptotic cells. Representative fields from thymi of control and Abl-T^−/− mice are shown in (A) (magnification: x20). A summary of numbers of activated caspase-3 positive cells from 9 mice, analyzed by the Ariol SL50 automated image analysis system (Applied Imaging Corp) of each genotype is shown in (B). Statistics: student T-test (one tailed) **p = 0.004. (C) Slides were also stained for TUNEL using the in situ cell death detection kit and viewed under the Olympus FV1000 microscope (x60) using Fluview1000V.1.5 software. (D) Extracts from freshly isolated thymocytes were analysed by Western blotting using antibodies to p38 Thr180/Tyr182, JNK Thr183/Tyr185, or GSK3α Tyr279/GSK3β Tyr216. Loading was checked by reprobing the membrane with antibodies to α-Tubulin.

Figure 4

Loss of Abl impairs mitogen-induced activation and proliferation of mature T cells. (A) Freshly isolated splenocytes from control or Abl-T^−/− mice were plated at equal numbers in the presence of ConA (5 μg/mL). After 72 hrs, viable cells were counted as described in Figure 1. Freshly isolated and ConA-activated splenocytes were subjected to flow cytometric analysis (staining for Thy1.2) to determine their T cell content. The initial numbers of T cells in control and Abl-T^−/− mice were taken as 100%. Data represent 11 mice from each genotype. Statistical analysis was performed by using the two-tailed student t test: ^§p < 0.0001. (B) Freshly isolated splenocytes from control or Abl-T^−/− mice were plated at equal numbers in the presence of anti-CD3 antibodies (1 μg/mL) plus IL-2 (10 U/mL) for 48 hrs. The numbers of viable T cells in freshly isolated and activated splenocytes were determined as described above. The initial number of T cells in control and Abl-T^−/− mice were taken as 100%. Data represent 6 mice from each genotype. Statistical analysis was performed by using the two-tailed student t test: §p < 0.0001. (C) 5 × 10⁵ T cells isolated from splenocytes using the EasySep Pan-T kit, were seeded on anti-CD3/anti-CD28 coated 24 wells in 1 ml medium. The numbers of live T cells were counted 8 times for each of 4 wells after 48 hrs and 72 hrs, and the average presented. (D) Splenocytes were activated as in (B) with anti-CD3 antibody (1 μg/mL) plus IL-2 (10 U/mL). After 48 hrs incubation, cells were pulsed with BrdU (20 μM) for 1 hr, stained with FITC-conjugated anti-BrdU (Dako Cytomation) and PI (5 μg/mL), and analyzed as in Figure 2B. Representative results are shown in (D) (dot plot), (E) (histogram), and a summary of 3 independent experiments with a total of 9 mice from each genotype is shown in (F). Statistical analysis was performed by using the one-tailed student t test:**p = 0.01. (G) Splenocytes were activated and pulsed with BrdU as described in (C). Cells were then washed and incubated in complete medium supplemented with IL-2 (10 U/mL), chased for 16 h, before they were harvested and treated as described in 2B. Representative results from 1 hr and 16 hrs chase are shown in (G), and a summary of 2 representative experiments with a total of 6 mice from each genotype is shown in (H). Data are presented as the % of dead cells among the BrdU⁺ cells. Statistical analysis was performed by using the one-tailed student t test: ***p value = 0.001 for 16 h chase. (I) Freshly isolated splenocytes from control or Abl-T^−/− mice were labeled with CFSE (5 μM) before activation with anti-CD3 antibody (1 μg/mL) plus IL-2 (10 U/mL). After 72 hrs of incubation, splenocytes were stained with APC-conjugated antibodies to CD4 or CD8, and the intensity of CFSE on CD4⁺ as well as CD8⁺ cells determined by FACS analysis. Representative data are shown in (I). The percentages of cells in each cell division were calculated (see Material and Methods) and are plotted in (J). Statistical analysis was performed by using the one-tailed student t test: p values *p < 0.05, **p < 0.01, ***p < 0.001, ^§p < 0.0001.

Figure 5

Loss of Abl causes a reduction in the levels of activation markers on mitogenically stimulated mature T cells. Lymphocytes from lymph nodes of control or Abl-T^−/− mice were activated with antibodies to CD3 (1.25 μg/mL) and CD28 (0.5 μg/mL). After 72 hrs, lymphocytes were stained with fluorochrome-conjugated antibodies to the following activation markers: APC-CD25, PE-CD69, PE-CD11a and FITC-CD71, and analyzed by FACS. Data are presented for all live cells (A), and for CD4⁺ cells (B). Graphs show percentages of positive cells, representing data from 5 mice of each genotype. Statistical analysis was performed by using the one-tailed student T test, p values *p < 0.05, **p < 0.01, ***p < 0.001, §p < 0.0001. (C) Equal number of isolated naïve T cells was subjected to RT-PCR analysis for IL-2 mRNA expression: T cells isolated from spleens using the EasySep Pan-T kit, were subjected to RT-PCR analysis for IL-2 mRNA levels. The expression levels of GAPDH mRNA were used to monitored the amount of mRNA used. Naïve splenic Abl-T^−/− cells showed lower basal IL-2 mRNA expression than control T cells.

Figure 6

Abl-T^−/− mice are unable to reject transplanted tumors. Cohorts of 8–10 weeks old control or Abl-T^−/− mice were injected intraperitoneally (i.p.) with a single inoculum of 3 × 10⁶ live PD1.6 thymic lymphoma cells expressing luciferase. At the indicated times after inoculation the mice were injected i.p. with luciferin (Promega) and the integrated light units were detected with a CCD camera and quantified by Metaview software. Levels of luminescence were summarized from all mice that developed tumors in the two experimental groups (n = 26 each group) (A) A graph presenting the average luminescence from all mice in each group as captured at indicated days. (B) Graph presents survival of the two groups of mice. Mice were sacrificed when moribund. All surviving mice were sacrificed at day 35 after injection for visual inspection of tumor growth. (C) Representative images of individual control mouse (top) and Abl-T^−/− (bottom) mouse taken at the indicated days. (D) In vitro killing assay: Control or Abl-T^−/− mice were immunized with 5 × 10⁶ PD1.6-Luc cells by i.p. injection. After 13 days the spleens were taken, and splenocytes were re-stimulated ex vivo with 5 × 10⁴ irradiated (10 Gy) PD1.6-Luc for 4 days in the presence of 30 U/ml IL-2 in 2 ml medium in 24-well plates. Thereafter, the re-stimulated splenocytes were incubated at various ratios with 1 × 10⁵ CFSE-labeled PD1.6-Luc cells in 200 microliter medium in 96-U well for 6 hrs, and percentage of PI-positive CFSE-positive cells were analysed on FACS. (F) At the indicated times after tumor transplantation sera were collected from the mice. The levels of PD1.6-Luc-specific antibodies in the sera were measured by staining PD1.6-Luc cells with the sera followed by secondary staining with Cy5-conjugated donkey anti-mouse IgG antibodies. The fluorescent intensity was monitored by FACS. Graphs present the percent of positively stained PD1.6-Luc cells. Sera from naïve mice (prior to tumor injection) were used as negative controls. Statistical analysis was performed by using the two-tailed student t test: §p < 0.0001.