Background/Purpose . The abundance of cytokines controls the length of immune responses through poorly defined mechanisms. B55β is a molecule that triggers apoptosis in activated T cells when cytokine levels decrease. T cells from a subset of patients with systemic lupus erythematosus (SLE) exhibit resistance to apoptosis that is associated to failure to express B55β. The aim of this study was to determine the molecular mechanisms through which B55β controls the survival of activated T cells.

Methods: Most experiments were performed in human primary T cells obtained from healthy donors. MCF7 cells were used in some experiments. T cells were activated with anti-CD3 and anti-CD28 (1 μg/mL). After day 3, fresh RPMI and IL-2 (100 U/mL) were replenished every 48 hours. Apoptosis was induced at day 10 by transferring the cells to fresh RPMI devoid of IL-2, in the presence of IL-2 blockade (anti-human IL-2). Apoptosis was quantified by flow cytometry (Annexin V/ propidium iodide staining). Forced expression and knockdown of B55β was induced by transducing activated T cells with lentiviruses encoding human B55β or B55β-specific shRNAs, respectively. Protein phosphorylation status was assessed by flow cytometry and Western blotting. Induction of pro-apoptotic genes was quantified by real time PCR.  

Results . Maximal expression of B55β (protein) was reached after 2 hours of IL-2 deprivation. This induction was associated to dephosphorylation of AKT S473 and T308 that occurred 4 hours after IL-2 withdrawal. IL-2 deprivation-induced AKT dephosphorylation was abrogated in T cells infected with lentivirus encoding B55β-specific shRNA. Accordingly, apoptosis was significantly reduced by B55β knockdown. Complementary experiments showed that forced expression of B55β caused apoptosis of activated T cells that was associated to dephosphorylation of AKT S473. Simultaneous forced expression of AKT annulled apoptosis induced by B55β indicating that B55β induces cell death through inactivation of AKT. To determine the effects of IL-2 deprivation on the PI3K-AKT-mTOR axis, we assessed the phosphorylation status of 18 members of the pathway using an intracellular signaling array. The results confirmed that AKT is dephosphorylated by IL-2 deprivation but that the pathway is not uniformly downregulated. mTORC1 and its substrates were mostly not affected whereas other AKT substrates, in particular the pro-apoptotic protein Bad and the transcription factors FoxO1 and 3a, were activated by dephosphorylation. Accordingly, induction of pro-apoptotic genes regulated by FoxO factors was observed. Further experiments confirmed that B55β overexpression activates the FoxO factors inducing the upregulation of pro-apoptotic genes, in particular Noxa, Puma and HRK.

Conclusion . B55β is a key regulator of AKT. Upon its induction by IL-2 deprivation, it causes AKT dephophorylation triggering programmed cell death through the induction and activation of the BH3-only pro-apoptotic proteins Bad, Noxa, Puma, and HRK. Our results demonstrate a novel mechanism of how AKT is regulated in activated T cells in response to cytokine abundance.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/b55%ce%b2-regulates-t-cell-survival-through-the-modulation-of-akt-during-cytokine-deprivation/