Bim controls IL-15 availability and limits engagement of multiple BH3-only proteins

Abstract

During the effector CD8+ T-cell response, transcriptional differentiation programs are engaged that promote effector T cells with varying memory potential. Although these differentiation programs have been used to explain which cells die as effectors and which cells survive and become memory cells, it is unclear if the lack of cell death enhances memory. Here, we investigated effector CD8+ T-cell fate in mice whose death program has been largely disabled because of the loss of Bim. Interestingly, the absence of Bim resulted in a significant enhancement of effector CD8+ T cells with more memory potential. Bim-driven control of memory T-cell development required T-cell-specific, but not dendritic cell-specific, expression of Bim. Both total and T-cell-specific loss of Bim promoted skewing toward memory precursors, by enhancing the survival of memory precursors, and limiting the availability of IL-15. Decreased IL-15 availability in Bim-deficient mice facilitated the elimination of cells with less memory potential via the additional pro-apoptotic molecules Noxa and Puma. Combined, these data show that Bim controls memory development by limiting the survival of pre-memory effector cells. Further, by preventing the consumption of IL-15, Bim limits the role of Noxa and Puma in causing the death of effector cells with less memory potential.

Figure 1

Bim is critical for the initial contraction of KLRG1^hiCD127^lo effector CD8⁺ T cells. Groups of WT and Bim^−/− mice (n=4–6 mice per genotype per time point) were infected intraperitoneally (i.p.) with 2 × 10⁵ p.f.u./mouse LCMV and killed 10, 15, 24 or 40 days post LCMV infection. Splenocytes were stained with D^b-GP33 tetramers and antibodies against KLRG1, CD127, CD8 and CD44 and analyzed by flow cytometry. D^b-GP33-specific CD8⁺ CD44⁺ cells were gated. (a) The graphs show the total numbers±S.E.M. of KLRG1^hiCD127^lo, KLRG1^hiCD127^hi or KLRG1^loCD127^hi subsets on indicated days after infection. (b) Representative dot plots for KLRG1 and CD127 are shown after gating on CD8⁺ GP33⁺ cells in each group on indicated days. (c) Graphs show percentages of CD8⁺ GP33⁺ KLRG1^hiCD127^lo or KLRG1^loCD127^hi subsets on indicated days after infection. Data are representative of two independent experiments. P values for statistically significant differences were calculated by Student's t-test and *P≤0.05 and **P≤0.01

Figure 2

KLRG1^loCD127^hi effector CD8⁺ T cells are enriched due to extrinsic factors in Bim^−/− mice. (a) As depicted in the figure, 5 × 10³ WT or Bim^−/− P14 Thy1.1⁺ CD8⁺ T cells were transferred into congenic WT or Bim^−/− mice (n=4–6 recipient mice per genotype per timepoint) and infected with LCMV 1 day later. (b and c) Graphs show percentages of Thy1.1⁺ P14 or Thy1.2⁺ endogenous cells within GP33⁺ CD8⁺ T cells on day 10 (b) or day 20 (c) after infection. (d and e) Graphs show percentages (d) or total numbers (e) of KLRG1^hiCD127^lo or KLRG1^loCD127^hi subsets within the total Thy1.1⁺ P14 cells analyzed on day 20 after infection. Data are representative of two independent experiments

Figure 3

Lack of Bim in dendritic cells does not impact survival of effector CD8⁺ T-cell subsets. (a) Splenocytes from Bim^f/f and CD11cCRE⁺ Bim^f/f mice were stained with antibodies against CD11c, CD11b and TCR and intracellularly against Bim. Histograms show staining for Bim (black) or isotype control (gray) in the indicated subsets from each group of mice. (b–d) Bone marrow from Rag^−/− Bim^f/f or Rag^−/−Cd11c⁻CRE⁺Bim^f/f (CD45.2⁺) mice were mixed with WT bone marrow (CD45.1⁺ CD45.2⁺) at 9:1 ratio and transferred into lethally irradiated WT (CD45.1⁺) congenic mice (n=6 recipient mice per genotype per time point). After 12–16 weeks of engraftment, mice were infected with LCMV and killed 10 or 24 days later. (b and c) CD45.1⁺ (host) or CD45.2⁺ (Rag^−/− CD11cCRE⁺ Bim^f/f) or CD45.1⁺ CD45.2⁺ (WT)-derived cells are gated. From these gates, TCR⁺, B220⁺ or TCR⁻ B220⁻ cells are gated. From B220⁻ TCR⁻ cells, CD11c⁺ cells are gated. (c) Histograms show staining for Bim (black) or isotype control (gray) in the B220-TCR-CD11c+, TCR+ or B220+ subsets indicated from each group of bone marrow chimeras. (d) Graphs show total numbers of CD8⁺ GP33⁺ KLRG1^hiCD127^lo or KLRG1^loCD127^hi subsets on indicated days after infection. Data are representative of two independent experiments

Figure 4

T-cell-specific loss of Bim increases survival of effector CD8⁺ T cells with preferential enrichment for KLRG1^loCD127^hi cells. (a) Splenocytes from Bim^f/f and dLckCRE⁺ Bim^f/f mice (n=4) were stained with antibodies against CD8, CD4 and TCR and intracellularly against Bim. Histograms show staining for Bim (black) or isotype control (gray) in subsets indicated from each group of mice. (b and c) Bim^f/f and dLckCre⁺ Bim^f/f mice (n=6–10) were infected with LCMV and killed 10 or 22 days later. (b) Graphs show total numbers of CD8⁺GP33⁺ KLRG1^hiCD127^lo and CD8⁺GP33⁺ KLRG1^loCD127^hi cells on indicated days after infection. (c) Graphs show total numbers of CD8⁺ GP33⁺ cells or percentages of CD8⁺ GP33⁺ KLRG1^hiCD127^lo or KLRG1^loCD127^hi subsets in indicated groups 22 days after infection. (d) 5 × 10³ WT or Bim^−/− P14 Thy1.1⁺ CD8⁺ T cells were transferred into congenic WT or Bim^−/− mice (n=4–6 recipient mice per genotype per timepoint) and infected with LCMV 1 day later. Graphs show total numbers of CD8⁺ CD44^hi GP33-sp cells (p14 and endogenous) versus overall CD8⁺ CD44^hi cells in different recipient animals on day 20 after infection. Data are representative of three independent experiments. **P≤0.01

Figure 5

IL-15 enhances survival of Bim-deficient KLRG1^hiCD127^lo effector CD8⁺ T cells. (a and b) Groups of WT or Bim^−/− mice (n=4 per genotype) were infected with LCMV and treated either with PBS or IL-15/IL-15Rα immune-complexes (IL-15-IC) on days 10, 12 and 14 p.i. and killed on day 15. Splenocytes were stained with D^bgp33 tetramers and antibodies against KLRG1, CD127 and CD8. Graphs show percentages (a) or numbers (b) of CD8⁺ GP33⁺ KLRG1^hiCD127^lo or KLRG1^loCD127^hi subsets on day 15 p.i. (c) Mice were also given BrDU the last 2 days before killing. Graph shows the percentages of BrDU⁺ cells within each subset. Data are representative of two independent experiments. Similar results were obtained in dLck-CreBim^f/f mice. **P≤0.01

Figure 6

IL-15 regulates survival of KLRG1^hiCD127^lo effector cells by redundant pro-apoptotic Bcl-2 family members. (a) 5 × 10³ WT or Bim^−/− P14 Thy1.1⁺ CD8⁺ T cells were transferred into congenic WT or IL-15^−/− (n=5–7) mice and infected with LCMV 1 day later. Graphs show percentages or numbers of KLRG1^hiCD127^lo or KLRG1^loCD127^hi subsets within the total Thy1.1⁺ P14 GP33⁺ CD8⁺ T cells analyzed on day 20 after infection. (b and c) Groups of WT, IL-15^−/−, IL-15^−/−-Bim^−/− and Bim^−/− mice (n=>10) were infected with LCMV and killed 10, 24 or 40 days later. Splenocytes were stained with H2D^b-gp33 tetramers and antibodies against KLRG1, CD127 and CD8. (b) Representative dot plots and percentages of each subset are shown after gating on CD8⁺ GP33⁺ cells in each group on day 24 p.i. (c) Graphs show total numbers of KLRG1^hiCD127^lo or KLRG1^loCD127^hi cells on the days indicated. (d) Groups of Bax^f/f Bak^−/− and dLckCre⁺ Bax^f/f Bak^−/− mice (n=7–10) were killed on day 10 or 22 after LCMV infection. Graphs show total numbers KLRG1^hiCD127^lo or KLRG1^loCD127^hi cells on the days indicated. Results are representative of two independent experiments. **P≤0.01

Figure 7

Bim, Noxa and Puma contribute to effector T-cell contraction. (a) Groups of C57BL/6 mice were infected with LCMV and on day 24 were killed and mRNA was isolated from sorted KLRG1^hiCD127^lo and KLRG1^loCD127^hi CD8⁺CD44^hi T cells. Results show the fold increase in Puma and Noxa mRNA in KLRG1^hiCD127^lo relative to KLRG1^loCD127^hi T cells as assessed by real-time RT-PCR. Cycle counts for Puma and Noxa were normalized to actin before assessment of fold increase. (b) Groups of C57BL/6 mice were infected with LCMV and on day 24 were killed and spleen cells were stained with MHC tetramers and antibodies against CD8, CD44, KLRG1, intracellularly with antibodies against Noxa and Puma. Results show the mean fluorescence intensity of the Noxa or Puma signal in pre-memory KLRG1^loCD127^hi (white bars) and effector KLRG1^hiCD127^lo T cells (dark bars) ±S.E.M. (c–f) Groups of WT, Puma^−/−, dLckCre⁺bim^f/f and Puma^−/−dLckCre⁺bim^f/f were infected with LCMV and then killed on days 10 and 24 after infection. (c) Dot plots show the levels of KLRG1 (y axis) and CD127 (x axis) within CD8⁺CD44^hiGP33-gated cells. (d) Graph shows the fold loss of KLRG1^hiCD127^lo cells between days 10 and 24. (e and f) Graphs show the total numbers of KLRG1^hiCD127^lo and KLRG1^loCD127^hi CD8⁺CD44^hi GP33-specific T cells ±S.E.M. from either WT, Puma^−/−, dLckCre⁺bim^f/f and Puma^−/−dLckCre⁺bim^f/f mice. (g–j) Groups of WT, Noxa^−/−, dLckCre⁺bim^f/f and Noxa^−/−dLckCre⁺bim^f/f were infected with LCMV and then killed on days 10 and 24 after infection. (g) Dot plots show the levels of KLRG1 (y axis) and CD127 (x axis) within CD8⁺CD44^hiGP33-gated cells and (h) graph shows the fold loss of KLRG1^hiCD127^lo cells between days 10 and 24. (i and j) Graphs show the total numbers of KLRG1^hiCD127^lo and KLRG1^loCD127^hi CD8⁺CD44^hi GP33-specific T cells ±S.E.M. from either WT, Noxa^−/−, dLckCre⁺bim^f/f and Noxa^−/−dLckCre⁺bim^f/f mice on days 10 and 24 after infection. Results are pooled from two to three independent experiments. *Significant difference as assessed by Student's t-test and P≤0.01