Fig 1.
miR-155 deficiency delays lymphoproliferative disease in LAT-KI mice.
(A) Photographs of spleens and axillary, brachial and inguinal lymph nodes from WT (C57BL/6), miR155-/-, LAT-KI and DM (LAT-KI/miR155-/-) mice. Ages of the mice were 9 wks (WT) and 8 wks (other three genotypes). Photographs are representative of 9 experiments. (B) Spleen weights of mice of the indicated genotypes over time. Each symbol represents an individual mouse. (C) CD4 and CD8 surface marker expression as measured by flow cytometry. Ages of the mice were 11 wks (WT), 9 wks (miR155-/-), 10 wk (LAT-KI), 10 wks (DM) and 19 wks (DM-old). The results are representative of 9 experiments. (D) Number of CD4+ T cells from axillary, brachial and inguinal lymph nodes of 8–10 wk old mice of the indicated genotypes. Each symbol represents an individual mouse.
Fig 2.
miR-155 deficiency results in increased basal CD4+ T cell activation and proliferation in LAT-KI mice.
(A) CD5 and CD69 surface marker expression as measured by flow cytometry in CD4+ lymph node T cells of mice of the indicated genotypes. Ages of the mice were the same as in Fig 1C. The results are representative of 9 experiments. (B) In vivo BrdU labeling. CD4+ lymph node T cells from mice previously injected with BrdU. Ages of the mice were 10 wks (WT) and 8 wks (other 3 genotypes). The results are representative of 3 experiments. (C) Negatively selected CD4+ T cells from the indicated genotypes were stimulated with αCD3ε/CD4 (10 μg/ml, 3 min.). Ages of the mice were 10 wks (WT, miR155-/-), 11 wks (LAT-KI), and 12 wks (DM). SDS whole cell lysates (WCLs) were analyzed by western blotting (WB). A representative WB is shown (n = 3).
Fig 3.
miR-155 deficiency results in increased basal apoptosis in LAT-KI T cells.
(A) Apoptotic (Annexin-V+/7-AAD-) lymph node CD4+ T cells as measured by flow cytometry. Ages of the mice were as in Fig 1C. The results are representative of 8 experiments. (B) FAS and FAS ligand surface expression as measured by flow cytometry in CD4+ lymph node T cells of mice of the indicated genotypes. Ages of the mice were as in Fig 1C. The results are representative of 3 experiments. (C) Negatively selected CD4+ T cells from the indicated genotypes were stimulated with αCD3ε/CD4 (10 μg/ml, 3 min). Ages of the mice were 11 wks (WT), 10 wks (miR155-/-), 11 wks (LAT-KI), and 12 wks (DM). SDS WCLs were analyzed by WB (n = 3). FL indicates full length, C3 Caspase 3 and C9 Caspase 9.
Fig 4.
miR-155 negatively regulates the transcription factor FOXO3, which positively regulates the pro-apoptotic factor BIM.
(A) Negatively selected CD4+ T cells from WT and LAT-KI mice were stimulated with αCD3ε/CD4 (10 μg/ml) for the indicated times. Ages of the mice were 11 wks (WT), 7 and 11 wks (LAT-KI young and old). SDS WCLs were analyzed by WB (n = 5). (B) CD4+ T cell purification, stimulation and WB were as in Fig 4A. Ages of the mice were 11 wks (WT, LAT-KI) and 14 wks (miR155-/-). SDS WCLs were analyzed by WB (n = 3). (C) CD4+ T cell purification, stimulation and WB were as in Fig 4A. Ages of the mice were 9 wks (WT), 12 wks (miR155-/-, LAT-KI), and 14 wks (DM). SDS WCLs were analyzed by WB (n = 3). (D) miR-155 was overexpressed in mouse CD4+ cells by retroviral infection. Mock infection was performed as a negative control. In both cases, GFP was expressed to identify infected cells. Sorted GFP+CD4+ T cells were stimulated with αCD3ε/CD4 (10 μg/ml) for 0, 3 or 10 min. SDS WCLs were analyzed by WB (n = 2).
Fig 5.
miR-155 deficiency decreases signaling in the PI3K/mTOR pathway.
Negatively selected CD4+ T cells from the indicated genotypes were stimulated with αCD3ε/CD4 (10 μg/ml, 3 min). Ages of the mice were 11 wks (WT), 10 wks (miR155-/-), 11 wks (LAT-KI), and 12 wks (DM). SDS WCLs were analyzed by WB (n = 3).
Fig 6.
miR-155 deficiency activates PAK1 and a PAK1-nucleated apoptotic pathway (JNK/FOXO3/BIM/Caspase 9).
(A) Negatively selected CD4+ T cells from the indicated genotypes were stimulated with αCD3ε/CD4 (10 μg/ml, 3 min.). Ages of the mice were 11 wks (WT), 10 wks (miR155-/-), 11 wks (LAT-KI), and 12 wks (DM). SDS WCLs were analyzed by WB (n = 3). (B) Jurkat T cells were transfected with Flag-PAK1 and PLC-γ1CI-HA cDNAs. 24h post-transfection, cells were treated for 4h with MEK inhibitor (U0126, 20 μM) or JNK inhibitor (SP600125, 20 μM). Lamin B-enriched nuclear and tubulin-enriched cytosolic fractions were prepared. PAK1/JNK-mediated FOXO3 nuclear translocation was studied by WB (n = 3). (C) Jurkat T cells were transfected with FOXO3a, BIM/Bcl2l11 or control siRNAs (150 nM). After 24h, cells were super-transfected with siRNAs (150 nM) plus PLC-γ1CI-HA, Flag-PAK1, or both cDNAs (10 μg). After an additional 36h, cells were lysed. Lysates (75%) were subjected to an active Caspase 9 IP and the 25% remaining lysates were used to make WCLs. Samples were analyzed by WB (n = 3).
Fig 7.
mTOR inhibition activates PAK1 signaling and a PAK1-nucleated apoptotic pathway (JNK/FOXO3/BIM/Caspase 9).
(A) Naturally SHIP-1-deficient Jurkat T cells were transfected with SHIP-1 cDNA (10 μg). 24h post-transfection, SDS WCLs were analyzed by WB(n = 4). (B) Jurkat T cells pre-treated with PI3K inhibitor (LY-294002, 50 μM) or control inactive enantiomer (LY-303511, 50 μM) for 90 min were stimulated with αCD3 (2 μg/ml) for the indicated times. SDS WCLs were analyzed by WB (n = 3). (C) Jurkat T cells were treated with Rapamycin or KU-0063794 (100 nM) for 0, 2, 4, or 6h. SDS WCLs were analyzed by WB (n = 3). (D) Jurkat T cells were transfected with Raptor/RPTOR, PAK1, both, or control siRNAs (200 μM). 48h post-transfection, SDS WCLs were analyzed by WB (n = 3). (E) Jurkat T cells were first starved (0.5% FCS) for 16h then pre-treated for 2h with cycloheximide (CHX, 50 μg/ml). After a 2h CHX pre-treatment, cells were not washed and Rapamycin (100 nM) was added to the media. Every hour SDS WCLs were made. Samples were analyzed by WB. Quantitation of a representative WB is shown (n = 3). The WB can be found in (B) of S4 Fig. (F) Jurkat T cells were transfected with Pak1 or control siRNAs (200 μM). 48h post-transfection, cells were treated with Rapamycin or KU-0063794 (100 nM, 2h). SDS WCLs were analyzed by WB (n = 4). (G) Jurkat T cells were transfected with FOXO3, BIM or control siRNAs (200 nM). After 36h, cells were treated with Rapamycin (100 nM, 16h) and lysed. Lysates (75%) were subjected to an active Caspase 9 IP and the 25% remaining lysates were used to make WCLs. Samples were analyzed by WB (n = 3).
Fig 8.
Proposed pathways mediating miR-155 control of lymphoproliferative disease in LAT-KI CD4+ T cells act through BIM.
(A) BIM deficiency accelerates lymphoproliferative disease in DM mice. Spleen weights of 6–10 wk old mice of the indicated genotypes (upper chart). Number of CD4+ + CD8+ T cells from axillary, brachial and inguinal lymph nodes of 6–10 wk old mice of the indicated genotypes (lower chart). Each symbol represents an individual mouse. (B) Proposed pathways mediating miR-155 control of lymphoproliferative disease in LAT-KI CD4+ T cells. Gray arrows indicate relative action caused by miR-155 deficiency. The function of AKT may be replaced by PDK1.