In T lymphocytes, the mitogen-activated protein kinase (MAPK) p38 regulates pleiotropic functions and is activated by canonical MAPK signaling or the alternative activation pathway downstream of the T cell antigen receptor (TCR). Here we found that senescent human T cells lacked the canonical and alternative pathways for the activation of p38 but spontaneously engaged the metabolic master regulator AMPK to trigger recruitment of p38 to the scaffold protein TAB1, which caused autophosphorylation of p38. Signaling via this pathway inhibited telomerase activity, T cell proliferation and the expression of key components of the TCR signalosome. Our findings identify a previously unrecognized mode for the activation of p38 in T cells driven by intracellular changes such as low-nutrient and DNA-damage signaling (an 'intrasensory' pathway). The proliferative defect of senescent T cells was reversed by blockade of AMPK-TAB1–dependent activation of p38.
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We thank S.S. Marelli for discussions. Supported by the Medical Research Council (A.L.), the Biotechnology and Biological Science Research Council (BB/J006750/1 to A.N.A. and S.M.H.) and the Instituto de Salud Carlos III, Spain (D.E.).
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Integrated supplementary information
(a) Immunoblot of total Lat, SLP-76 and PLC-γ 1 expression in freshly isolated human CD4+ CD27/CD28 defined subsets. GAPDH was used as a loading control. (b) The relative expression of Lat, SLP-76 and PLC-γ 1 vs. total GAPDH in the CD4+ CD27/CD28 defined subsets from 3 different individuals. (c) Representative overlay and (d) pooled Fluo-4 AM data from 3 independent experiments showing intracellular calcium influx after αCD3 activation (10 μg/mL, 5’) in CD27+ CD28+ and CD27- CD28- CD4+ T cells, as measured by flow-cytometry. In (b) a one-way analysis of variance (ANOVA) for repeated-measures with a Bonferroni post-test correction. In (d) a paired Student’s t test. *p< 0,05, **p<0.01, and ***p< 0.001. Error bars depict s.e.m
Supplementary Figure 2 Confirmation of the knockdown of AMPKα and TAB1 by lentiviral vector in senescent CD27–CD28– CD4+ T cells.
(a) Extended map and (b) schematic representation of the p-Siren HIV lentiviral vector system used for gene knock-down. The shRNA of interest was cloned downstream the U6-promoter between the BamHI and EcoRI restriction sites. Measurement of (c) AMPKα and (d) TAB1 expression by both immunoblot (top) and quantitative PCR (bottom) in highly differentiated CD27- CD28- CD4+ T cells transduced with either shAMPKα or shTAB1, as indicated. An irrelevant scrambled short hairpin (shCtrl) expressing vector was used as internal control. Immunoblots are representative of 3 independent experiments. Samples for quantitative PCR were analyzed from 4 different donors and normalized against housekeeping GAPDH. Knockdown efficiency was evaluated 96 hours post-transduction (day 7). **p<0.01 and ***p< 0.001 values were assessed by a paired Student’s t test. Errors bars depict s.e.m.
Supplementary Figure 3 Enhanced levels of ROS endogenously activate ATM, AMPK and p38 in senescent CD27–CD28– CD4+ T cells.
(a) Intracellular ROS levels by Dihydroethidium (DHE) in CD27+ CD28+ and CD27- CD28- CD4+ T cells directly ex vivo, as measured by flow cytometry. Data are representative of 4 independent experiments. (b) Representative overlays and (c) pooled phospho-flow data from 3 different donors showing the effect of the ROS scavenger superoxide-dismutase (SOD; 100 U, 60’) on AMPK (Thr172), p38 (Thr180,Tyr182) and ATM (Ser1921) phosphorylation in senescent CD27- CD28- CD4+ T cells. All *p< 0,05, **p<0.01, and ***p< 0.001 values were calculated by a paired Student’s t test. Error bars depict s.e.m
Supplementary Figure 4 Silencing p38 restores telomerase and proliferative activities in senescent human CD27–CD28– CD4+ T cells.
(a) Experimental design. Purified human senescent CD27- CD28- CD4+ T cells were activated by αCD3 plus rh-IL-2, and then transduced 48 hours later with lentiviral particles. A second hit transduction was performed 24 hours later. Following transduction, medium containing rh-IL-2 was replaced every 2-3 days. CD27- CD28- CD4+ T cells were maintained in activation medium and analyzed for functional readouts at the indicated time points (see text). For long-term cultures, transduced cells were subjected to a number of two subsequent rounds of activation by αCD3 plus rh-IL2, every 10 days. (b) Measurement of p38 expression by both immunoblot (left) and quantitative PCR (right) in CD27- CD28- CD4+ T cells, transduced as indicated. Immunoblots are representative of 2 independent experiments. Samples for quantitative PCR were analyzed from 3 different donors and normalized against housekeeping GAPDH. Knockdown efficiency was evaluated 96 hours post-transduction (day 7). (c) Telomerase activity by TRAP assay in CD27- CD28- CD4+ T cells transduced as indicated. Experiments were performed from 3 separate donors, 96 hours post-transduction. Proliferative activity by either (d) 3H thymidine incorporation or (e) dye dilution assay in CD27- CD28- CD4+ T cells, transduced as indicated. Experiments were performed from 3 separate donors, 96 hours after transduction. (f) The pooled results of 3 separate experiments performed as in (e). (g) Replicative lifespan by cumulative PDs of long-term cultured CD4+ CD27- CD28- T cells, transduced as indicated. Cumulative PDs were assessed by absolute cell number enumeration from 3 separate donors. All *p< 0,05, **p<0.01, and ***p< 0.001 values were calculated by a paired Student’s t test. Error bars depict s.e.m.
Supplementary Figure 5 p38-mediated modulation of cell-cycle machinery in senescent human CD27–CD28– CD4+ T cells.
Representative overlay and pooled phospho-flow data (n=3) showing the effect of the p38 inhibitor BIRB (500 nM) on (a,b) p27, (c,d) D1 cyclin and (e,f) under-phosphorylated Rb expression in CD27- CD28- CD4+ T cells, activated by CD3/rh-IL-2 for 36 hours. (g) Rb inactivation and (h) p53 activation were assessed by measuring the ratio of the inhibitory phosphorylated Rb (Ser807,Ser811) to under-phosphorylated Rb and phosphorylation of p53 at residue Ser46, respectively. All data are from 3 independent experiments. All **p<0.01 values were calculated by a paired Student’s t test. Error bars depict s.e.m
Measurement of (a) telomerase activity by TRAP assay and (b) proliferation by thymidine incoporation in relatively undifferentiated CD27+ CD28+ CD4+ T cells activated by αCD3/CD28 antibodies and transduced with either shCtrl or shP38, in the presence of DMSO control or the AMPK agonist A-769662 (150 μM). (c) Representative Facs plot and (d) pooled data of Ki67 expression by CD27+ CD28+ CD4+ T cells activated by CD3/CD28 in the presence or absence of the p38 inhibitor BIRB 796 (500 nM), under glucose starvation. Activated CD27+ CD28+ CD4+ T cells grown in the presence of glucose [20mM] served as positive proliferation control. (e) Telomerase activity by TRAP assay in glucose starved CD27+ CD28+ CD4+T cells activated for 36 hours in the presence or absence of BIRB 796. In (a,b) *p< 0,05, **p<0.01, and ***p< 0.001 values were calculated by a one-way analysis of variance (ANOVA) for repeated-measures with a Bonferroni post-test correction; in (d,e) *p< 0,05 was calculated by a paired Student’s t test. Error bars depict s.e.m.
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Lanna, A., Henson, S., Escors, D. et al. The kinase p38 activated by the metabolic regulator AMPK and scaffold TAB1 drives the senescence of human T cells. Nat Immunol 15, 965–972 (2014). https://doi.org/10.1038/ni.2981
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