The proteome and transcriptome of stress granules and P bodies during human T lymphocyte activation.

Stress granules (SGs) and processing bodies (PBs) are membraneless cytoplasmic assemblies regulating mRNAs under environmental stress such as viral infections, neurological disorders, or cancer. Upon antigen stimulation, T lymphocytes mediate their immune functions under regulatory mechanisms involving SGs and PBs. However, the impact of T cell activation on such complexes in terms of formation, constitution, and relationship remains unknown. Here, by combining proteomic, transcriptomic, and immunofluorescence approaches, we simultaneously characterized the SGs and PBs from primary human T lymphocytes pre and post stimulation. The identification of the proteomes and transcriptomes of SGs and PBs indicate an unanticipated molecular and functional complementarity. Notwithstanding, these granules keep distinct spatial organizations and abilities to interact with mRNAs. This comprehensive characterization of the RNP granule proteomic and transcriptomic landscapes provides a unique resource for future investigations on SGs and PBs in T lymphocytes.

Regulatory Mechanisms of Inhibitory Immune Checkpoint Receptors Expression.

T cells responding to persistent tumor or viral antigens progressively lose their functional properties, a feature known as exhaustion. This state is also characterized by cell-surface expression of multiple inhibitory immune checkpoint receptors (IRs). Cancer immunotherapy by immune checkpoint targeting has shown impressive clinical outcomes, but requires substantial improvement given the limited number of patients who benefit from the treatment. Targeting the mechanisms controlling immune checkpoint expression could represent a step towards this aim. Accumulating data indicate that this strategy can limit immune checkpoint expression, in some instances simultaneously inhibiting several immune checkpoints. This review discusses various mechanisms through which IRs are activated or regulated, and ways these mechanisms could be exploited to develop more effective future immunotherapies.

Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

Despite the clinical success of blocking inhibitory immune checkpoint receptors such as programmed cell death-1 (PD-1) in cancer, the mechanisms controlling the expression of these receptors have not been fully elucidated. Here, we identify a post-transcriptional mechanism regulating PD-1 expression in T cells. Upon activation, the PDCD1 mRNA and ribonucleoprotein complexes coalesce into stress granules that require microtubules and the kinesin 1 molecular motor to proceed to translation. Hence, PD-1 expression is highly sensitive to microtubule or stress granule inhibitors targeting this pathway. Evidence from healthy donors and cancer patients reveals a common regulation for the translation of CTLA4, LAG3, TIM3, TIGIT, and BTLA but not of the stimulatory co-receptors OX40, GITR, and 4-1BB mRNAs. In patients, disproportionality analysis of immune-related adverse events for currently used microtubule drugs unveils a significantly higher risk of autoimmunity. Our findings reveal a fundamental mechanism of immunoregulation with great importance in cancer immunotherapy.

Radiation-induced mitotic cell death and glioblastoma radioresistance: a new regulating pathway controlled by integrin-linked kinase, hypoxia-inducible factor 1 alpha and survivin in U87 cells.

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1 alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity. In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.

Autoimmunity during thymectomy-induced lymphopenia: role of thymus ablation and initial effector T cell activation timing in nonobese diabetic mice.

Autoimmune diseases develop in selected normal mouse strains when thymectomy (Tx) is performed at 3 days of age (d3-Tx). Insufficient T cell regulation after Tx may result from a defect in regulatory T (Treg) cells or from an augmented effector T (Teff) cell number/pathogenicity. We have previously shown that Tx at 3 wk (wk3-Tx), the age of massive islet Ag release, accelerates diabetes onset. We now have determined diabetes incidence in d3-Tx nonobese diabetic mice and compared the frequency and function of their Teff and Treg cells with those of wk3-Tx mice. We found that d3-Tx had no effect on diabetes incidence, but induced gastritis. After day 3 and week 3 Tx, Treg cells were fully competent and their frequency increased. The number of diabetogenic T cells was greatly amplified after wk3-Tx and likely overcame Treg cell control, leading to an early tolerance breakdown. By contrast, in d3-Tx mice, activation concerned few cells and Teff cell amplification remained controlled. This suggests that Tx enhances autoimmunity when it coincides with the first encounter of autoreactive T cells with their cognate Ag. The relationship between Tx-induced lymphopenia, tissue remodeling, and autoimmunity is discussed.

Modulating estrogen receptor-related receptor-alpha activity inhibits cell proliferation.

High expression of the estrogen receptor-related receptor (ERR)-alpha in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRalpha reduces the proliferation of various cell lines and blocks the G(1)/S transition of the cell cycle in an ERRalpha-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21(waf/cip)(1) at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice.

The orphan receptor ERRalpha interferes with steroid signaling.

The estrogen receptor-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily that has been shown to interfere with the estrogen-signaling pathway. In this report, we demonstrate that ERRalpha also cross-talks with signaling driven by other steroid hormones. Treatment of human prostatic cells with a specific ERRalpha inverse agonist reduces the expression of several androgen-responsive genes, in a manner that does not involve perturbation of androgen receptor expression or activity. Furthermore, ERRalpha activates the expression of androgen response elements (ARE)-containing promoters, such as that of the prostate cancer marker PSA, in an ARE-dependent manner. In addition, promoters containing a steroid response element can be activated by all members of the ERR orphan receptor subfamily, and this, even in the presence of antisteroid compounds.

Potentiation of ICI182,780 (Fulvestrant)-induced estrogen receptor-alpha degradation by the estrogen receptor-related receptor-alpha inverse agonist XCT790.

ICI182,780 (Fulvestrant) is a pure anti-estrogen used in adjuvant therapies of breast cancer. This compound not only inhibits the transcriptional activities of the estrogen receptor-alpha (ER alpha) but also induces its proteasome-dependent degradation. The latter activity is believed to be required for the antiproliferative effects of ICI182,780. Estrogen receptor-related receptor-alpha (ERR alpha) is an orphan member of the nuclear receptor superfamily that is expressed in a wide range of tissues including breast tumors, in which its high expression correlates with poor prognosis. Although not regulated by any natural ligand, ERR alpha can be deactivated by the synthetic molecule XCT790. Here we demonstrate that this compound also induces a proteasome degradation of ERR alpha. We also show that although it does not act directly on the steady-state level of ER alpha, XCT790 potentiates the ICI182,780-induced ER alpha degradation. We suggest that treatment with XCT790 could thus enhance the efficacy of ICI182,780 in ER alpha-dependent pathologies such as breast cancer.

Interleukin-7 induces apoptosis of 697 pre-B cells expressing dominant-negative forms of STAT5: evidence for caspase-dependent and -independent mechanisms.

The transcription factors STAT5A and STAT5B (STAT: signal transducer and activator of transcription) play a major role in the signaling events elicited by a number of growth factor and cytokine receptors. In this work, we aimed to investigate the role of STAT5 in human precursor B cell survival by introducing dominant-negative (DN) forms of STAT5A or STAT5B in the 697 pre-B cell line. All clones expressing DN forms of either transcription factor exhibited a higher spontaneous apoptotic rate that was massively enhanced upon interleukin-7 (IL-7) stimulation. This was associated with caspase 8 cleavage, mitochondrial transmembrane potential disruption and caspase 3 activation. However, the DN forms of STAT5 did not alter the expression of Bcl-2, Bax, Bcl-x, Bim, A1 and Mcl1 proteins in IL-7-stimulated cells. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromylmethyl ketone partially suppressed IL-7-mediated mitochondrial transmembrane potential disruption and cell death, suggesting that IL-7 induced the death of DN STAT5 expressing 697 cells through caspase-dependent and -independent mechanisms that both require mitochondrial activation.

TGF-beta1 modulates Fas (APO-1/CD95)-mediated apoptosis of human pre-B cell lines.

We have previously shown that Fas-induced apoptosis is markedly enhanced by IL-7 in human pre-B but not pro-B cell lines. In addition, pre-B cell receptor (pre-BCR) ligation significantly potentiates the IL-7 effects on Fas-triggered pre-B cell death. We show herein that transforming growth factor (TGF)-beta 1 sharply reduces Fas-induced death rate of pre-B but not pro-B cells. TGF-beta 1 causes inhibition of Fas-mediated disruption of mitochondrial transmembrane potential and cleavage of caspase 8, Bid and caspase 3. Bcl2 expression is markedly increased in TGF-beta 1-treated pre-B cells, whereas cellular FLICE-like inhibitory protein long (c-FLIPL), Bcl-XL, Bax, and Bad expression remains unchanged. TGF-beta 1 causes a selective growth arrest of pre-B cells in G0/G1 phase of the cell cycle and induces a partial down-modulation of both Fas and pre-BCR expression. All TGF-beta 1-mediated effects, but Bcl2 up-regulation, can be reproduced by the LY294002 phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor but not by inhibitors of the MAPK/ERK (MEK) and Janus kinase (Jak)/STAT pathways, which promote cell death. Akt phosphorylation is strongly inhibited by TGF-beta1 in pre-B but not pro-B cells and is not modified by Fas engagement. Altogether, our findings suggest that TGF-beta1 prevents Fas-induced apoptosis of pre-B lines by inhibiting PI3K pathway and by enhancing expression of Bcl2. They also suggest that the PI3K/Akt pathway is involved in the control of Fas and pre-BCR expression, a checkpoint in B cell development.