Loss of T cell receptor-induced Bmi-1 in the KLRG1(+) senescent CD8(+) T lymphocyte

Abstract

Clones of CD8(+) T cells specific for viral antigens must avoid replicative senescence to maintain continuous production of new effector cells during chronic viral infections. In the present study, we have determined whether this capability may be related to Bmi-1, a transcriptional repressor that is required for the maintenance of hematopoietic stem cells and certain neural stem cells and that mediates its antisenescence function by inhibiting transcription of the Ink4a/Arf tumor suppressor locus. Ligation of the T cell receptor increased the levels of Bmi-1 mRNA and protein in primary CD8(+) T cells. The increased expression was reversible upon removal of antigen but could be maintained by using stimulation with the IL-2 receptor. Specific suppression of Bmi-1 by using a lentivirally encoded short hairpin RNA inhibited the proliferation of IL-2-stimulated CTLL-2 cytotoxic T cells and primary CD8(+) T cells. Ectopically expressed Bmi-1 enhanced the expansion of primary CD8(+) T cells stimulated by IL-2 and IL-7 in vitro and by homeostatic signals in vivo. Taken together, these findings indicate that Bmi-1 is required for CD8(+) T cell clonal expansion and is positively regulated by receptors that mediate this response. Therefore, the observation that the ability of the T cell receptor to induce Bmi-1 is maintained in the subset of replication-competent, antigen-experienced CD8(+) T cells that do not express the killer cell lectin-like receptor G1 (KLRG1) but is developmentally switched off in the senescent, KLRG1(+) subset suggests that Bmi-1 is a molecular determinant of the capacity of a CD8(+) T cell clone to persist during chronic viral infections.

Fig. 1.

TCR signaling and induction of Bmi-1 expression in CD8⁺ T cells. (a) Naïve OT-I T cells were cultured for 24 h in medium alone or in medium containing incremental concentrations of SIINFEKL peptide and assessed by flow cytometry for Bmi-1 expression after staining with isotype control (black line) or anti-Bmi-1 antibody (red line). (b) Whole-cell lysates of naïve OT-I cells were generated 0 and 20 h after culturing cells in medium containing 2.5 nM SIINFEKL peptide and were assessed by immunoblot for Bmi-1 and β-actin. (c and d) Naïve OT-I cells were cultured in the presence of 2.5 nM SIINFEKL peptide for various lengths of time and then assessed for Bmi-1 expression by flow cytometry after staining with isotype control (black line) or anti-Bmi-1 antibody (red line) (c) and assessed for Bmi-1 mRNA by quantitative RT-PCR (d). The mean fluorescent intensity for specific Bmi-1 staining is represented by the number in each histogram. These experiments were performed multiple times with comparable results.

Fig. 2.

Maintenance of Bmi-1 expression by IL-2. Naïve OT-I cells were cultured for 24 h in medium with 2.5 nM SIINFEKL peptide. The cells were then transferred to fresh medium alone or medium containing incremental concentrations of IL-2 and cultured for an additional 20 h, after which Bmi-1 expression was assessed by staining with isotype control (black line) or anti-Bmi-1 antibody (red line). The mean fluorescent intensity for specific Bmi-1 staining is represented by the number in each histogram. The experiments shown were performed multiple times with comparable results.

Fig. 3.

Inhibition of proliferation of CD8⁺ T cells by a lentivirus expressing an shRNA specific for Bmi-1. CTLL-2 cells maintained with IL-2 were infected at an moi of 5 with lentiviral vectors expressing EGFP alone (pll3.7) or together with a Bmi-1-specific shRNA (pll3.7-shBmi-1). (a) Whole-cell lysates of CTLL-2 cells (lane 1), cells infected with pll3.7 (lane 2), or cells infected with pll3.7-shBmi-1 (lane 3) were subjected to immunoblot analysis with antibodies against Bmi-1 and β-actin. (b) CTLL-2 cells infected with either the pll3.7 or pll3.7-shBmi-1 lentiviral vector were assessed for Bmi-1 and Mel-18 mRNA by using quantitative RT-PCR. (c) The number of CTLL-2 cells infected with pll3.7 (open squares) or pll3.7-shBmi-1 (filled squares) was measured during continuous culture in the presence of IL-2. (d) CTLL-2 cells were transduced with a retroviral vector expressing human CD2 alone (circles) or together with Bmi-1 containing silent mutations at the shRNA binding site (triangles). Transduced cells were superinfected with either pll3.7 (open triangles) or pll3.7-shBmi-1 (filled triangles and filled circles), and the number of EGFP⁺, lentivirally transduced cells was assayed periodically during growth in the presence of IL-2. (e) The number of EGFP⁺ OT-I cells infected with pll3.7 (open squares) or pll3.7-shBmi-1 (filled squares) was measured during continuous culture in the presence of IL-2 and IL-7. The experiments shown were performed multiple times with comparable results.

Fig. 4.

Enhanced proliferation in vitro of CD8⁺ T cells expressing ectopic Bmi-1. (a) OT-I cells that had been activated with anti-CD3ε in the presence of IL-2 and IL-7 were transduced with pMig/Thy1 or with pMig/Thy1/Bmi-1 and maintained for 6 d in the presence of IL-2 and IL-7. On day 4 after transduction, cells expressing rat Thy1 were purified, and lysates were subjected to analysis by Western blot with antibodies specific for Bmi-1, HA, and β-actin. (b) In a replicate experiment, OT-I cells were transduced with pMig/Thy1 (open squares) or pMig/Thy1/Bmi-1 (filled squares) and assessed for growth in the presence of IL-2 and IL-7. The experiments shown were performed multiple times with comparable results, except for the Western blot, which was performed once.

Fig. 5.

Enhanced homeostatic expansion of CD8⁺ T cells expressing ectopic Bmi-1. Rat Thy1⁺ OT-I cells (4 × 10⁵) that had been transduced with pMig/Thy1 or pMig/Thy1/Bmi-1 during proliferation in the presence of IL-2 and IL-7 were adoptively transferred to Rag2^−/− recipients. After 215 d, the number of rat Thy1⁺ CD8⁺ T cells in the blood, spleen, femur, and liver was measured, with recovery from the latter two sites being normalized by counting Gr-1⁺ cells. The means and SE are shown for each determination. The experiment has been repeated with similar results. *, P = 0.043 (paired Student's t test).

Fig. 6.

Impaired TCR-mediated induction of Bmi-1 expression in senescent KLRG1⁺ CD8⁺ T cells. (a) Naïve CD44^low KLRG1⁻ CD8⁺ T cells and antigen-experienced, CD44^high CD8⁺ T cells that were either KLRG1⁻ or KLRG1⁺ were purified from the spleens of four mice 8–9 weeks after infection with γMHV-M3-OVA virus and were either immediately assessed for expression of Bmi-1 or stimulated with anti-CD3ε, IL-2, and IL-7 for 24 h. Representative histograms demonstrate staining with isotype control (black lines) or anti-Bmi-1 antibody (red lines) of purified cells of a single mouse. The bar graphs to the right represent the mean ± SE of specific mean fluorescent intensity for Bmi-1 staining of cells from all four mice. (b) Stimulation of cells with anti-CD3ε, IL-2, and IL-7 was continued for an additional 16 h, with BrdU being added during the last 4 h. The bar graphs represent the mean ± SE of the percentage of cells that had incorporated BrdU. (c) Additional replicate samples of cells received Brefeldin A at time 0, when stimulation was initiated, and were then assessed at 3 h for intracellular IFN-γ. The bar graphs represent the mean ± SE of the percentage of cells that had intracellular IFN-γ. *, KLRG-1⁺ cells are significantly different from naïve and CD44^high KLRG-1⁻ cells: P = 0.0036 (a) and P = 0.0015 (b) (paired Student's t test).