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. 2005 Jan 17;201(2):259-66.
doi: 10.1084/jem.20042020. Epub 2005 Jan 10.

The Pim kinases control rapamycin-resistant T cell survival and activation

Casey J Fox  1 , Peter S Hammerman, Craig B Thompson
Affiliations

The Pim kinases control rapamycin-resistant T cell survival and activation

Casey J Fox et al. J Exp Med. .

Abstract

Although Pim-1 or Pim-2 can contribute to lymphoid transformation when overexpressed, the physiologic role of these kinases in the immune response is uncertain. We now report that T cells from Pim-1(-/-)Pim-2(-/-) animals display an unexpected sensitivity to the immunosuppressant rapamycin. Cytokine-induced Pim-1 and Pim-2 promote the rapamycin-resistant survival of lymphocytes. The endogenous function of the Pim kinases was not restricted to the regulation of cell survival. Like the rapamycin target TOR, the Pim kinases also contribute to the regulation of lymphocyte growth and proliferation. Although rapamycin has a minimal effect on wild-type T cell expansion in vitro and in vivo, it completely suppresses the response of Pim-1(-/-)Pim-2(-/-) cells. Thus, endogenous levels of the Pim kinases are required for T cells to mount an immune response in the presence of rapamycin. The existence of a rapamycin-insensitive pathway that regulates T cell growth and survival has important implications for understanding how rapamycin functions as an immunomodulatory drug and for the development of complementary immunotherapeutics.

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Figures

Figure 1.
Figure 1.
IL-4– and IL-7–dependent survival is intact in Pim-deficient T cells. (A) C57BL/6 splenic T cells were cultured without cytokine (−), with IL-4 (+), or IL-7 (+) for 3, 6, or 12 h (hr). Control was prepared from nonstimulated cells at the start of the culture period (0 h). Total cell lysates were serially probed for the expression of β-actin (Actin) as a loading control, Pim-1, or Pim-2 by Western blot. Due to alternative translational initiation, a single murine pim-1 transcript gives rise to two proteins of 34 (bottom band) and 44 kD (top band). Murine pim-2 encodes three isoforms of 34 (bottom band), 37 (middle band), and 40 kD (top band). The results shown are representative of three independent experiments. (B) T cells from Pim-1+/+2+/+ and Pim-1−/−2−/− littermates (key) were cultured without cytokine (None), IL-4 (+ IL-4), or IL-7 (+ IL-7) and the percent of viable cells measured daily for 4 d. Error bars represent standard deviation of the mean of triplicate samples. Results have been reproduced in five additional experiments.
Figure 2.
Figure 2.
IL-4 and IL-7 promote rapamycin-independent survival. (A) Lysates were prepared from nonstimulated C57BL/6 T cells (0 h) or after 3 or 12 h of IL-4 (+) or IL-7 (+) treatment and serially probed for actin, Pim-1, Pim-2, phospho-Serine 473-Akt (pSer473 Akt), and Total Akt. Results shown were reproduced in four additional experiments. (B) C57BL/6 T cells were preincubated in the presence (+) or absence (−) of 50 nM rapamycin (RAPA) for 1 h before stimulation with IL-4 (+) or IL-7 (+) for 30 or 90 min. Control lysate was from nonstimulated T cells (time 0). Lysates were serially probed for actin, phospho-serine 371-p70 S6 Kinase (pSer371 p70S6K), Total p70S6K, phospho-Serine 235/Serine 236-S6 (pSer235/236 S6), and Total S6 protein by Western blot. Results shown represent two experiments. (C) Viability was measured daily for 3 d in Pim-1+/+2+/+ T cells cultured without treatment, cytokine − RAPA, or cytokine + RAPA (keys). (Top) IL-4. (Bottom) IL-7. Error bars represent standard deviation of the mean of triplicate samples and summarize six individual experiments. (D) Pim-1 and Pim-2 induction are rapamycin resistant. C57BL/6 T cells were stimulated with IL-4 (+) or IL-7 (+) in the presence (+) or absence (−) of 50 nM RAPA for 3 or 12 h. Control was prepared from nonstimulated T cells (time 0). Lysates were serially probed for actin, Pim-1, and Pim-2.
Figure 3.
Figure 3.
Pim-2 deficiency confers rapamycin sensitivity. (A) Viability was measured daily for 3 d in splenic T cells from Pim-1+/+2+/+, Pim-1−/−2+/+, Pim-1+/+2−/−, and Pim-1−/−2−/− littermates cultured without cytokine (None), + IL-4, or + IL-7 in the presence (+) or absence (−) of 25 nM RAPA (key). Error bars represent standard deviation of the mean of triplicate samples. Data have been reproduced in eight additional experiments. (B) T cells from female Pim-1−/−2−/− and Pim-1+/+2+/+ littermates were stimulated with IL-7 (+) in the presence (+) or absence (−) of 50 nM RAPA and lysates prepared at 1/2 and 12 h. Control was from nonstimulated cells prepared at the start of the experiment (0 h). Lysates were serially probed for the expression of actin, Bcl-2, Bcl-xL, Bim, Bad, phospho-serine 136 Bad (pSer136 Bad), and phospho-serine 112 Bad (pSer112 Bad). For Bim, the upper band corresponds to BimEL at ∼23 kD and the lower to BimL at 16 kD. Results have been reproduced in three independent experiments.
Figure 3.
Figure 3.
Pim-2 deficiency confers rapamycin sensitivity. (A) Viability was measured daily for 3 d in splenic T cells from Pim-1+/+2+/+, Pim-1−/−2+/+, Pim-1+/+2−/−, and Pim-1−/−2−/− littermates cultured without cytokine (None), + IL-4, or + IL-7 in the presence (+) or absence (−) of 25 nM RAPA (key). Error bars represent standard deviation of the mean of triplicate samples. Data have been reproduced in eight additional experiments. (B) T cells from female Pim-1−/−2−/− and Pim-1+/+2+/+ littermates were stimulated with IL-7 (+) in the presence (+) or absence (−) of 50 nM RAPA and lysates prepared at 1/2 and 12 h. Control was from nonstimulated cells prepared at the start of the experiment (0 h). Lysates were serially probed for the expression of actin, Bcl-2, Bcl-xL, Bim, Bad, phospho-serine 136 Bad (pSer136 Bad), and phospho-serine 112 Bad (pSer112 Bad). For Bim, the upper band corresponds to BimEL at ∼23 kD and the lower to BimL at 16 kD. Results have been reproduced in three independent experiments.
Figure 4.
Figure 4.
The Pim kinases confer rapamycin-resistant T cell blastogenesis. (A) C57BL/6 splenic T cells were activated by αCD3/αCD28 and lysates were prepared after 0, 1/2, 1, 2, 3, 6, and 12 h of stimulation and serially probed for the expression of actin, Pim-1, and Pim-2 proteins by Western blot. (B) Activation-induced p70S6K and S6 phosphorylation is rapamycin sensitive. C57BL/6 splenic T cells were preincubated for 1 h in the presence (+) or absence (−) of 50 nM RAPA and activated with αCD3/αCD28 for 0, 10, and 30 min. Lysates were probed for actin, phospho-serine 371-p70 S6 kinase (pSer371 p70S6K), total p70S6K, phospho-serine 235/serine 236-S6 (pSer235/236 S6), and total S6 expression. (C) In the experiments described in B, Western blots were also performed with lysates from rapamycin-treated and control cells after 3 and 12 h of activation and probed for actin, Pim-1, and Pim-2 expression. (D) Splenic T cells from Pim-1+/+2+/+, Pim-1−/−2+/+, Pim-1+/+2−/−, and Pim-1−/−2−/− mice were activated by αCD3/αCD28 in the absence (−) or presence (+) of 25 nM RAPA (key). Control cells were cultured with αCD28 only (αCD28). After 2 d of culture, forward scatter, and surface CD69, CD25, and CD62L expression was assessed. (E) Cell cycle was assessed in the Pim-1+/+2+/+ and Pim-1−/−2−/− T cells described in D by BrdU incorporation analysis. The percentage of cells in the G1 (2N-PI+BrdU−; bottom left), S (PI+BrdU+; top), and G2M (4N-PI+BrdU−; bottom right) phases of the cell cycle is shown (insets). Results shown are representative of six experiments. (F)5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester–labeled input T cells from Pim-1+/+2+/+ or Pim-1−/−2−/− mice were activated for 4 d in the presence (+) or absence (−) of 25 nM RAPA. Control cells were cultured with αCD28 only for 2 d (αCD28) and showed no decay in fluorescence relative to input cells at day 0. Numbers above the leftmost brackets represent the percent of cells that had divided more than three generations. For Pim-1−/−2−/− cells, the majority of the input population failed to divide (*). Data are representative of three experiments.
Figure 4.
Figure 4.
The Pim kinases confer rapamycin-resistant T cell blastogenesis. (A) C57BL/6 splenic T cells were activated by αCD3/αCD28 and lysates were prepared after 0, 1/2, 1, 2, 3, 6, and 12 h of stimulation and serially probed for the expression of actin, Pim-1, and Pim-2 proteins by Western blot. (B) Activation-induced p70S6K and S6 phosphorylation is rapamycin sensitive. C57BL/6 splenic T cells were preincubated for 1 h in the presence (+) or absence (−) of 50 nM RAPA and activated with αCD3/αCD28 for 0, 10, and 30 min. Lysates were probed for actin, phospho-serine 371-p70 S6 kinase (pSer371 p70S6K), total p70S6K, phospho-serine 235/serine 236-S6 (pSer235/236 S6), and total S6 expression. (C) In the experiments described in B, Western blots were also performed with lysates from rapamycin-treated and control cells after 3 and 12 h of activation and probed for actin, Pim-1, and Pim-2 expression. (D) Splenic T cells from Pim-1+/+2+/+, Pim-1−/−2+/+, Pim-1+/+2−/−, and Pim-1−/−2−/− mice were activated by αCD3/αCD28 in the absence (−) or presence (+) of 25 nM RAPA (key). Control cells were cultured with αCD28 only (αCD28). After 2 d of culture, forward scatter, and surface CD69, CD25, and CD62L expression was assessed. (E) Cell cycle was assessed in the Pim-1+/+2+/+ and Pim-1−/−2−/− T cells described in D by BrdU incorporation analysis. The percentage of cells in the G1 (2N-PI+BrdU−; bottom left), S (PI+BrdU+; top), and G2M (4N-PI+BrdU−; bottom right) phases of the cell cycle is shown (insets). Results shown are representative of six experiments. (F)5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester–labeled input T cells from Pim-1+/+2+/+ or Pim-1−/−2−/− mice were activated for 4 d in the presence (+) or absence (−) of 25 nM RAPA. Control cells were cultured with αCD28 only for 2 d (αCD28) and showed no decay in fluorescence relative to input cells at day 0. Numbers above the leftmost brackets represent the percent of cells that had divided more than three generations. For Pim-1−/−2−/− cells, the majority of the input population failed to divide (*). Data are representative of three experiments.
Figure 4.
Figure 4.
The Pim kinases confer rapamycin-resistant T cell blastogenesis. (A) C57BL/6 splenic T cells were activated by αCD3/αCD28 and lysates were prepared after 0, 1/2, 1, 2, 3, 6, and 12 h of stimulation and serially probed for the expression of actin, Pim-1, and Pim-2 proteins by Western blot. (B) Activation-induced p70S6K and S6 phosphorylation is rapamycin sensitive. C57BL/6 splenic T cells were preincubated for 1 h in the presence (+) or absence (−) of 50 nM RAPA and activated with αCD3/αCD28 for 0, 10, and 30 min. Lysates were probed for actin, phospho-serine 371-p70 S6 kinase (pSer371 p70S6K), total p70S6K, phospho-serine 235/serine 236-S6 (pSer235/236 S6), and total S6 expression. (C) In the experiments described in B, Western blots were also performed with lysates from rapamycin-treated and control cells after 3 and 12 h of activation and probed for actin, Pim-1, and Pim-2 expression. (D) Splenic T cells from Pim-1+/+2+/+, Pim-1−/−2+/+, Pim-1+/+2−/−, and Pim-1−/−2−/− mice were activated by αCD3/αCD28 in the absence (−) or presence (+) of 25 nM RAPA (key). Control cells were cultured with αCD28 only (αCD28). After 2 d of culture, forward scatter, and surface CD69, CD25, and CD62L expression was assessed. (E) Cell cycle was assessed in the Pim-1+/+2+/+ and Pim-1−/−2−/− T cells described in D by BrdU incorporation analysis. The percentage of cells in the G1 (2N-PI+BrdU−; bottom left), S (PI+BrdU+; top), and G2M (4N-PI+BrdU−; bottom right) phases of the cell cycle is shown (insets). Results shown are representative of six experiments. (F)5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester–labeled input T cells from Pim-1+/+2+/+ or Pim-1−/−2−/− mice were activated for 4 d in the presence (+) or absence (−) of 25 nM RAPA. Control cells were cultured with αCD28 only for 2 d (αCD28) and showed no decay in fluorescence relative to input cells at day 0. Numbers above the leftmost brackets represent the percent of cells that had divided more than three generations. For Pim-1−/−2−/− cells, the majority of the input population failed to divide (*). Data are representative of three experiments.
Figure 5.
Figure 5.
Pim-1 and Pim-2 are required for rapamycin-resistant T cell expansion and activation in vivo. (A) 6–8-wk-old Pim-1+/+2+/+ or Pim-1−/−2−/− mice (key) were pretreated with RAPA (+ RAPA) or no drug (− RAPA) before TSST (+ TSST) or vehicle (− TSST) injection. The percent of CD25+ peripheral T cells (gated on live Thy1+ cells) is shown. Error bars represent the standard error from the mean of 3 to 12 individuals in each group. (B) The percent of the T cells described in A that were positive for TCR Vβ3 is also shown.
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