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Cited by 88T-cell and B-cell ImmunologyAutophagy in Disease and TherapyPsoriasis: Treatment and Pathogenesis

Selective degradation of PU.1 during autophagy represses the differentiation and antitumour activity of TH9 cells

Open access
Rivera Vargas, Thaiz, Cai, Zhijian, Shen, Yingying, Dosset, Magalie, Benoit-Lizon, Isis, Martin, Tiffany, Roussey, Aurélie, Flavell, Richard A. +2 more·2017·Nature Communications
DOI 10.1038/s41467-017-00468-wPMC PMC5602674
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Abstract

Abstract Autophagy, a catabolic mechanism that involves degradation of cellular components, is essential for cell homeostasis. Although autophagy favours the lineage stability of regulatory T cells, the contribution of autophagy to the differentiation of effector CD4 T cells remains unclear. Here we show that autophagy selectively represses T helper 9 (TH9) cell differentiation. CD4 T cells lacking Atg3 or Atg5 have increased interleukin-9 (IL-9) expression upon differentiation into TH9 cells relative to Atg3- or Atg5-expressing control cells. In addition, the TH9 cell transcription factor, PU.1, undergoes K63 ubiquitination and degradation through p62-dependent selective autophagy. Finally, the blockade of autophagy enhances TH9 cell anticancer functions in vivo, and mice with T cell-specific deletion of Atg5 have reduced tumour outgrowth in an IL-9-dependent manner. Overall, our findings reveal an unexpected function of autophagy in the modulation of TH9 cell differentiation and antitumour activity, and prompt potential autophagy-dependent modulations of TH9 activity for cancer immunotherapy.

AI snapshot

Written from the paper’s full text.

TL;DR

Autophagy selectively restrains TH9 differentiation by targeting the transcription factor PU.1 for p62-dependent selective autophagic degradation. Blocking autophagy genetically or pharmacologically increases IL-9 production and enhances TH9 antitumour activity in mouse melanoma and colon cancer models.

Key findings

  • Atg5-deficient CD4 T cells showed a fivefold increase in IL-9 secretion upon TH9 differentiation by ELISA; Atg3- or Map1lc3b-deficient CD4 T cells also had enhanced IL-9, while IFN-γ and IL-17 secretion were not significantly affected and TH2 cytokines were only modestly induced without significant IL-4/IL-5 increases.
  • PU.1 was K63-ubiquitinated and degraded through p62-dependent selective autophagy; PU.1 half-life increased from 3 h to more than 6 h in Atg5-deficient TH9 cells, and p62 inhibition enhanced both PU.1 expression and IL-9 secretion.
  • Metformin prevented and chloroquine enhanced TH9 differentiation in a dose-dependent manner; chloroquine also enhanced IL-9 secretion from human TH9 cells differentiated from three healthy blood donors.
  • Chloroquine-treated TH9 cells had enhanced anticancer effects in vivo, and Atg5fl/fl*CD4-Cre mice showed reduced B16-OVA and MC38 tumour growth in an IL-9-dependent manner, with anti-IL-9 antibody abrogating the benefit.

Methods

  • Mouse genetic models included Atg5fl/fl*CD4-Cre, Atg3fl/flER-Cre, Map1lc3b−/−, IL-9-EGFP, OT-II and TRP-1 TCR transgenic mice; naive CD4+CD62LhiCD44lo T cells were differentiated into TH0, TH1, TH2, TH9, TH17 and Treg subsets with plate-bound anti-CD3/anti-CD28 and polarizing cytokines.
  • Human naive CD4 T cells from three healthy blood donors were differentiated into TH9 cells with TGF-β and IL-4, with or without chloroquine.
  • Analyses included ELISA, qRT-PCR, flow cytometry, western blot, immunoprecipitation, proximity ligation assay, mass spectrometry, pull-down assays, cycloheximide/DRB treatments, and in vivo B16-OVA, B16F10 and MC38 tumour models with adoptive transfer and anti-IL-9 neutralization; statistics used Student’s t-test and ANOVA.

Limitations

  • The paper states that the results “do not rule out any autophagy-independent effects of metformin and chloroquine on TH9 cell survival and proliferation,” and that tumour-derived factors affecting TH9 cell induction remain to be fully characterized.
  • The paper notes that regulation of TH9 cell development in the tumour microenvironment is still incompletely understood and that the findings “may begin to explain” low TH9 abundance in tumours.
  • Evident from the text: human data are limited to three healthy blood donors, with one representative donor shown for the chloroquine dose response; in vivo antitumour evidence is from mouse models, and chloroquine itself altered TH9 proliferation and apoptosis, complicating interpretation of its effects.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41467-017-00468-w

Cites

56 of 56 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

25 of 39 cited sources checked · 13 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Protein expression of GATA-3 was unaffected by metformin or chloroquine.

    “the protein expression of the transcription factor GATA-3, which is essential for TH9 cell differentiation28, was unaffected by metformin or chloroquine”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [28] Dardalhon, V. et al. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+IL-10+Foxp3(−) effector T cells. Nat. Immunol. 9, 1347–1355 (2008). source

      The citation attaches to the statement that GATA-3 is essential for TH9 differentiation, not to the citing authors’ metformin or chloroquine result. In Results (IL-4 inhibition of Foxp3 induction; page unavailable), GATA-3-deficient cells failed to produce IL-9 and IL-10 under the tested differentiation conditions. The Discussion (page unavailable) cautions that this may be an indirect effect and reports that the resulting IL-9–IL-10-producing cells did not express GATA-3, so the source does not establish that GATA-3 is essential for their differentiation. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    TH9 cells harbour superior anticancer properties compared to those of TH1 or TH17 cells upon adoptive transfer in vivo.

    “We and others have shown that TH9 cells harbour superior anticancer properties compared to those of TH1 or TH17 cells upon adoptive transfer in vivo33, 34.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [33] Vegran, F. et al. The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells. Nat. Immunol. 15, 758–766 (2014). source
    • cites [34] Purwar, R. et al. Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells. Nat. Med. 18, 1248–1253 (2012). source
  • Premisecitation

    PU.1 is an ETS family transcription factor induced by TGF-β during TH9 cell differentiation.

    “PU.1 is an ETS family transcription factor that is induced by transforming growth factor-β (TGF-β) during TH9 cell differentiation”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [40] Chang, H. C. et al. The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation. Nat. Immunol. 11, 527–534 (2010). source

      In Results (‘PU.1 promotes the development of IL-9–secreting T cells’; page unavailable), TH9 cells had more Sfpi1 mRNA than TH2 cells, but the authors say only that Sfpi1 ‘might be a TGF-β-induced gene.’ The claim presents TGF-β induction as established. The ETS-family designation appears in the abstract, not in the body passages used for this verdict. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    PU.1 is essential for TH9 cell generation.

    “is essential for TH9 cell generation as illustrated by the inability of PU.1-deficient CD4+ T cells to produce IL-9 upon TH9 cell differentiation40.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [40] Chang, H. C. et al. The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation. Nat. Immunol. 11, 527–534 (2010). source

      The Results section (‘PU.1 promotes the development of IL-9–secreting T cells’; page not supplied) reports decreased IL-9 secretion and expression in PU.1-deficient cells cultured under TH9 conditions. This supports PU.1’s importance for the TH9 phenotype, but not the citing sentence’s stronger assertion that those cells were unable to produce IL-9. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    Truncation of the PU.1 PEST domain did not lead to protein stabilization.

    “it is noteworthy that truncation of the PU.1 PEST domain did not lead to protein stabilization41”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [41] Nishiyama, C. et al. Functional analysis of PU.1 domains in monocyte-specific gene regulation. FEBS Lett. 561, 63–68 (2004). source

      The Discussion reports that the PEST-deleted PU.1 mutant had a lower protein level than wild-type PU.1 by Western blot, supporting the observation that deletion did not increase protein accumulation. It does not report a protein-stability or degradation-rate measurement, so the specific claim about stabilization is not directly established. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 22
  • May combine two claims. 25
  • The quote only partly states this claim. 3

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-9 favours CD8 T-cell activation in a model of pulmonary melanoma in mice.

    “IL-9 has been shown to favour CD8 T-cell activation in a model of pulmonary melanoma in mice36.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [36] Lu, Y. et al. Th9 cells promote antitumor immune responses in vivo. J. Clin. Invest. 122, 4160–4171 (2012). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Atg16l1-deficient TH2 cells have increased survival.

    “Atg16l1-deficient TH2 cells have increased survival.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [43] Kabat, A. M. et al. The autophagy gene Atg16l1 differentially regulates Treg and TH2 cells to control intestinal inflammation. eLife 5, e12444 (2016). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The amino acid sensor GCN2 controls gut inflammation through autophagy induction in CD11c antigen-presenting cells as well as epithelial cells, resulting in repression of TH17 cell responses.

    “this is exemplified by the ability of the amino acid sensor GCN2 to control gut inflammation through autophagy induction in CD11c antigen-presenting cells as well as epithelial cells, resulting in the repression of TH17 cell responses49.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [49] Ravindran, R. et al. The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature 531, 523–527 (2016). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Atg16l1-deficient Treg cells showed significant decreased survival compared to WT Treg cells.

    “They found that while Atg16l1-deficient Treg cells showed significant decreased survival compared to WT Treg cells”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [43] Kabat, A. M. et al. The autophagy gene Atg16l1 differentially regulates Treg and TH2 cells to control intestinal inflammation. eLife 5, e12444 (2016). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Autophagy affects the stability of Treg cells by coupling environmental cues and metabolic homeostasis.

    “Chi and co-workers16 recently showed that autophagy affects the stability of Treg cells by coupling environmental cues and metabolic homeostasis.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [16] Wei, J. et al. Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis. Nat. Immunol. 17, 277–285 (2016). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    GATA-3 expression is essential for TH9 but not for Treg cell differentiation.

    “One important observation is that, like in TH2 cells, GATA-3 expression is essential for TH9 but not for Treg cell differentiation28.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [28] Dardalhon, V. et al. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+IL-10+Foxp3(−) effector T cells. Nat. Immunol. 9, 1347–1355 (2008). source

      In Results (‘IL-4 inhibition of Foxp3-induction depends on STAT6’; page unavailable), GATA-3-deficient cells could not produce IL-9 or IL-10 under the tested differentiation conditions. Results (‘Relationship between Foxp3 and GATA-3’; page unavailable) also show increased TGF-β-induced Foxp3 expression without GATA-3, supporting the Treg part. But the Discussion says the IL-9–IL-10-producing cells did not express GATA-3 and that the knockout effect may be indirect, so the paper does not establish that GATA-3 expression is essential in TH9 cells themselves. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TH2 cells display an increased glycolytic rate.

    “In contrast to Treg cells, TH2 cells were shown to display an increased glycolytic rate44.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [44] Michalek, R. D. et al. Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets. J. Immunol. 186, 3299–3303 (2011). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Autophagy deficiency in Treg cells upregulates metabolic regulators and glycolysis, thus impairing Treg cell survival.

    “Autophagy deficiency in Treg cells upregulates metabolic regulators and glycolysis, thus impairing Treg cell survival16.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [16] Wei, J. et al. Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis. Nat. Immunol. 17, 277–285 (2016). source

      The Introduction and Results report increased mTORC1 and c-Myc activity, elevated glycolysis, and impaired survival in Atg7-deficient Treg cells. The Discussion links excess glycolysis to impaired lineage stability, while the Results link mTORC1 signaling to increased apoptosis; the paper does not establish that elevated glycolysis itself impairs survival. Our copy provides no page numbers for these passages. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In humans, polymorphisms in several autophagy-related genes (IRGM, LRRK2, SMURF1, ATG16L1) leading to autophagy deficiency are linked to IBD susceptibility.

    “In humans, polymorphisms in several autophagy-related genes, including IRGM, LRRK2, SMURF1 and ATG16L1, leading to autophagy deficiency are linked to inflammatory bowel disease (IBD) susceptibility50.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [50] Liu, J. Z. et al. Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations. Nat. Genet. 47, 979–986 (2015). source

      The Results section, “Biological implications of newly associated IBD loci” (page unavailable), identifies ATG16L1 and IRGM as Crohn’s disease susceptibility genes and links autophagy to its pathogenesis. The cited passages do not establish the claimed links for LRRK2 or SMURF1, or show that the associated variants cause autophagy deficiency. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Autophagy differentially regulates Treg and TH2 cells to control intestinal inflammation.

    “For instance, Kabat et al.43 elegantly reported how autophagy differentially regulates Treg and TH2 cells to control intestinal inflammation.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [43] Kabat, A. M. et al. The autophagy gene Atg16l1 differentially regulates Treg and TH2 cells to control intestinal inflammation. eLife 5, e12444 (2016). source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Expression of Gata3 is linked to induction of glycolysis after TCR activation in T cells and orchestrates essential metabolic adaptations enforced by autophagy deficiency.

    “Meanwhile, expression of Gata3 is linked to induction of glycolysis after TCR activation in T cells and orchestrates essential metabolic adaptations enforced by autophagy deficiency45.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [45] Wan, Y. Y. GATA3: a master of many trades in immune regulation. Trends Immunol. 35, 233–242 (2014). source

      In “Distinct aspects of T cell activation and proliferation require GATA3” (page unavailable), the review reports that TCR stimulation regulates GATA3 and that GATA3 loss prevents Myc up-regulation; it proposes a role for the GATA3–Myc axis in T-cell metabolism. It does not establish induction of glycolysis or describe metabolic adaptations caused by autophagy deficiency. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    TH9 cells have potent anticancer properties, especially upon adoptive transfer into melanoma-bearing mice.

    “Subsequently, this CD4 T-cell subset has gained further interest because of its potent anticancer properties, especially upon adoptive transfer into melanoma-bearing mice33, 34, 38, 39.”
    Quote in the paperSubstantial claim2 of 4 sources checked
    • cites [33] Vegran, F. et al. The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells. Nat. Immunol. 15, 758–766 (2014). source
    • cites [34] Purwar, R. et al. Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells. Nat. Med. 18, 1248–1253 (2012). source
    • cites [38] Nakatsukasa, H. et al. The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+) T cells. Nat. Immunol. 16, 1077–1084 (2015). source
    • cites [39] Kim, I. K. et al. Glucocorticoid-induced tumor necrosis factor receptor-related protein co-stimulation facilitates tumor regression by inducing IL-9-producing helper T cells. Nat. Med. 21, 1010–1017 (2015). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Protein inclusions observed in animal cells with defective autophagy are enriched in ubiquitin.

    “Protein inclusions observed in the animal cells with defective autophagy are enriched in ubiquitin22.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010). source

      The 'Degradation of LC3 and Selective Substrates' section says p62 accumulates in autophagy-deficient cells, and the References list mentions inclusion bodies in autophagy-deficient mice. Neither passage says those inclusions are enriched in ubiquitin; a search for ubiquitin found no passage establishing that point. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    TH9 cells are proinflammatory cells that contribute to tissue inflammation and defence against parasites.

    “TH9 cells were initially coined by Kuchroo and Stockinger laboratories as proinflammatory cells that contribute to tissue inflammation and defence against parasites28, 37.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [28] Dardalhon, V. et al. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+IL-10+Foxp3(−) effector T cells. Nat. Immunol. 9, 1347–1355 (2008). source

      The Discussion (page not provided) identifies IL-9+IL-10+Foxp3− T cells as proinflammatory and reports that they promote tissue inflammation. It does not link these cells to defence against parasites; the paper’s parasite-related references concern IL-10 and other immune responses. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [37] Veldhoen, M. et al. Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat. Immunol. 9, 1341–1346 (2008). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TH9 transcription factor PU.1 and IL-9 expression are crucial for the development of experimental colitis in two different mouse models.

    “Moreover, TH9 transcription factor PU.1 and IL-9 expression were shown to be crucial for the development of experimental colitis in two different mouse models28, 53.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [28] Dardalhon, V. et al. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+IL-10+Foxp3(−) effector T cells. Nat. Immunol. 9, 1347–1355 (2008). source

      The Results (Function of the IL-9+IL-10+Foxp3− T cells; page unavailable) show that IL-9-producing T cells induced colitis and worsened it when co-transferred with effector T cells in a mouse T-cell-transfer model. The Discussion explicitly says it was unclear whether IL-9 itself played a role. The paper does not establish that PU.1 or IL-9 expression is crucial for colitis, or report that finding in two mouse models. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • cites [53] Gerlach, K., McKenzie, A. N., Neurath, M. F. & Weigmann, B. IL-9 regulates intestinal barrier function in experimental T cell-mediated colitis. Tissue Barriers 3, e983777 (2015). source

      The Results (page unavailable) show that IL-9-deficient mice were almost completely protected from TNBS colitis and had fewer PU.1-expressing T cells. They do not establish that PU.1 itself is crucial for colitis development. The Discussion (page unavailable) mentions a second mouse model, but says a previous study found that Th9 cells prevented colitis in that model, not that they were crucial to its development. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    PU.1 undergoes K63 ubiquitination and degradation through p62-dependent selective autophagy.

    “In addition, the TH9 cell transcription factor, PU.1, undergoes K63 ubiquitination and degradation through p62-dependent selective autophagy.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PU.1 is the TH9 cell master transcription factor encoded by Sfpi1.

  • May combine two claims.

    Findingown data

    Atg5 deficiency modestly induces IL-4, IL-5 and IL-13 from TH2 cells without reaching a significant increase by ELISA analysis of IL-4 and IL-5 cytokines.

    “modestly induced IL-4, IL-5 and IL-13 from TH2 cells without reaching a significant increase by enzyme-linked immunosorbent assay (ELISA) analysis of IL-4 and IL-5 cytokines”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    TH9 cells treated with chloroquine show a significant increase of apoptosis and a reduced proliferation compared to control cells.

    “TH9 cells treated with chloroquine show a significant increase of apoptosis and a reduced proliferation compared to control cells”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    p62 expression was induced in TH9 cells and peaked 16 h after the initiation of differentiation.

    “Western blot analysis showed that p62 expression was induced in TH9 cells and peaked 16 h after the initiation of differentiation”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Chloroquine enhanced IL-9 secretion and increased the frequency and mean fluorescence intensity of IL-9-expressing cells.

    “a similar series of studies performed with chloroquine yielded opposite results, confirming that autophagy blockade enhances TH9 cell differentiation”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Similar results (reduced tumour growth and abrogation by anti-IL-9) were noted in the MC38 colon adenocarcinoma tumour model.

    “Similar results were noted in the MC38 colon adenocarcinoma tumour model (Fig. 6d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Naive CD4 T cells differentiated into TH9 cells in the presence of metformin or chloroquine respectively featured repressed or enhanced levels of PU.1 protein compared to controls.

    “naive CD4 T cells differentiated into TH9 cells in the presence of metformin or chloroquine respectively featured repressed or enhanced levels of PU.1 protein compared to controls”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PU.1 is the TH9 cell master transcription factor encoded by Sfpi1.

  • May combine two claims.

    Findingown data

    Metformin did not affect cell death of differentiating TH9 cells but reduced their proliferation.

    “Metformin however did not affect cell death of differentiating TH9 cells but reduced their proliferation”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Chloroquine, despite its effects on TH9 cell proliferation and apoptosis, markedly enhances TH9 cell IL-9 secretion on a per-cell basis, ultimately resulting in enhanced IL-9 secretion from chloroquine-treated TH9 cells.

    “Thus, chloroquine, despite its effects on TH9 cell proliferation and apoptosis, markedly enhances TH9 cell IL-9 secretion on a per-cell basis, ultimately resulting in enhanced IL-9 secretion from chloroquine-treated TH9 cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Metformin decreased IL-9 secretion measured by ELISA and quantitative PCR and reduced the frequency and mean fluorescence intensity of IL-9-expressing cells 3 days following differentiation initiation.

    “Differentiation of naive CD4 T cells obtained from IL-9-EGFP mice into TH9 cells in the presence of metformin decreased IL-9 secretion measured by ELISA and quantitative PCR (qPCR) (Fig. 3c) and both reduced the frequency and mean fluorescence intensity of IL-9-expressing cells 3 days following differentiation initiation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Absence of Atg5 promotes PU.1 protein stability, enhancing its half-life from 3 to more than 6 h.

    “Indeed, the absence of Atg5 promotes PU.1 protein stability, enhancing its half-life from 3 to more than 6 h”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PU.1 is the TH9 cell master transcription factor encoded by Sfpi1.

  • May combine two claims.

    Findingown data

    In WT TH9 cells, PU.1 is mainly nuclear with an important nuclear expression from 8 to 48 h of differentiation.

    “In WT TH9 cells, PU.1 is mainly nuclear with an important nuclear expression from 8 to 48 h of differentiation”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is the TH9 cell master transcription factor encoded by Sfpi1.

  • May combine two claims.

    Findingown data

    Metformin and chloroquine respectively prevented and enhanced TH9 cell differentiation in a dose-dependent manner.

    “We found that metformin and chloroquine respectively prevented and enhanced TH9 cell differentiation in a dose-dependent manner”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DRB efficiently prevented Sfpi1 gene expression but failed to block the chloroquine-induced enhanced expression of PU.1 protein.

    “We found that, while DRB efficiently prevented Sfpi1 gene expression, it failed to block the chloroquine-induced enhanced expression of PU.1 protein”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    UBA truncation significantly impaired p62 ability to recruit PU.1 without affecting its interaction with LC3-II.

    “we found that UBA truncation significantly impaired p62 ability to recruit PU.1 without affecting its interaction with LC3-II”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PU.1 is the TH9 cell master transcription factor encoded by Sfpi1.

  • The quote only partly states this claim.

    Findingown data

    Genetic or pharmacological blockade of autophagy in TH9 cells enhances anticancer functions in vivo in both established colon cancer and melanoma.

    “but also in vivo in both established colon cancer and melanoma.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Through its UBA domain, p62 is able to bind ubiquitinated PU.1 and interacts with LC3-II.

    “We also found that, through its UBA domain, p62 is able to bind ubiquitinated PU.1 and interacts with LC3-II.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Autophagy induction in TH9 cells reduces PU.1 stability and represses TH9 cell differentiation.

    “Our findings that autophagy induction in TH9 cells reduces PU.1 stability and represses TH9 cell differentiation”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    Differential expression of factors such as GATA-3 may lead to different metabolic responses in the absence of autophagy in CD4 T cells and explain why TH9 cells behave like TH2 cells regarding glycolysis.

    “Thus, differential expression of factors such as GATA-3 may lead to different metabolic responses in the absence of autophagy in CD4 T cells and explain why TH9 cells behave like TH2 cells regarding glycolysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Enhanced glycolytic metabolism used by TH2 cells makes them resistant to the metabolic changes triggered by autophagy deficiency.

    “This enhanced glycolytic metabolism used by TH2 cells makes them resistant to the metabolic changes triggered by autophagy deficiency.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    In the tumour microenvironment, TH9 cells undergo increased autophagy leading to decreased IL-9 expression.

    “Our results suggest that in the tumour microenvironment TH9 cells undergo increased autophagy leading to decreased IL-9 expression.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TH9 cells have an important function in driving UC.

    “Strikingly, recent studies have shown an important function of TH9 cells in driving UC.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [51] Gerlach, K. et al. TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells. Nat. Immunol. 15, 676–686 (2014). source
    • cites [52] Nalleweg, N. et al. IL-9 and its receptor are predominantly involved in the pathogenesis of UC. Gut 64, 743–755 (2015). source

  • May combine two claims.

    Interpretationown data

    During TH9 cell differentiation, PU.1 is K63-ubiquitinated and recruited by p62, which targets PU.1 for degradation through selective autophagy.

    “During TH9 cell differentiation, the TH9 cell master transcription factor PU.1 is K63-ubiquitinated and recruited by p62, which targets PU.1 for degradation through selective autophagy.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Lung tumour foci were enumerated in a blinded fashion.

    “Lung tumour foci were enumerated after 14 days in a blinded fashion.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All transgenic mice used were on a C57BL/6 background and were age-matched with WT controls for experiments.

    “All transgenic mice used were on a C57BL/6 background and were age-matched with WT controls for experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    All cells were routinely tested for mycoplasma contamination and found negative.

    “All cells were routinely tested for mycoplasma contamination using Mycoalert Mycoplasma Detection Kit (Lonza, LT07-318) and found negative.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Isolated naive T cells were routinely 98% pure.

    “Isolated naive T cells were routinely 98% pure.”
    Quote in the paperNot a substantial claim
42 of 42

  • The paper relies on this without support.

    PU.1 is the TH9 cell master transcription factor encoded by Sfpi1.

    “the TH9 cell master transcription factor encoded by Sfpi1 gene”

  • The paper relies on this without support.

    The regulation of TH9 cell development in the tumour microenvironment is incompletely understood.

    “However, the regulation of TH9 cell development in the tumour microenvironment is still incompletely understood.”

  • The paper relies on this without support.

    The tumour-derived factors affecting TH9 cell induction remain to be fully characterized.

    “While the tumour-derived factors affecting TH9 cell induction remain to be fully characterized”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • K63-ubiquitination of PU.1 promotes its degradation in a p62-dependent selective autophagy manner in Th9 cells, leading to reduced protein turnover and antitumor immunity.

    “Rivera Vargas and colleagues, recently demonstrated K63-ubuqtination of PU.1 promoted its degradation in a p62-depenent selective autophagy manner in Th 9 cells leading to reduced protein turnover and antitumor immunity (49).”

    From FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1 (2021) · cites this paper as [49]

    The Discussion (page unavailable) states that K63-ubiquitinated PU.1 is recruited by p62 for selective autophagic degradation in TH9 cells, and that blocking autophagy enhances their anticancer function. The claim's 'reduced protein turnover' does not follow: degradation increases PU.1 turnover; blocking that degradation stabilizes PU.1. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Different TRP-1 TH9 cell doses reported for the same experiment

    Major

    The Fig. 6b legend reports 0.1×106 TRP TH9 cells per mouse, whereas the corresponding tumour-growth method reports 5×104 cells-half that dose.

  • Immunoprecipitation input aliquot exceeds the starting lysate

    Minor

    The method starts with 1 ml of lysate but says 20 ml of the pre-cleared lysate was retained as input. That aliquot cannot be taken from the stated starting volume.

2 of 2

Cites

Flagged first, then by citations

2 of the 58 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Rivera Vargas, T., Cai, Z., Shen, Y., Dosset, M., Benoit-Lizon, I., Martin, T., Roussey, A., Flavell, R. A., Ghiringhelli, F., & Apetoh, L.
Affiliations
  • Zhejiang University (CN)
  • Inserm (FR)
  • Howard Hughes Medical Institute (US)
Corresponding author
Ghiringhelli, François
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-09-15
Volume
8
Issue
1
Pages
559-559
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-017-00468-w
PMC
PMC5602674
OpenAlex
W2754508153
Cited by
88
References
58
Citations by year
2026: 2 · 2025: 3 · 2024: 12 · 2023: 8 · 2022: 6 · 2021: 17 · 2020: 14 · 2019: 8 · 2018: 12 · 2017: 2
FWCI
3.93
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • HAL (Le Centre pour la Communication Scientifique Directe) (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Life Sciences › Immunology and Microbiology › Immunology › Psoriasis: Treatment and Pathogenesis
Keywords
autophagy, Th9 cells, PU.1, selective autophagy, K63-linked ubiquitination, p62, ATG5, ATG3
MeSH terms
Autophagy-Related Proteins, Autophagy-Related Protein 5, Proto-Oncogene Protein Spi-1, Animals, Autophagy, Cell Differentiation, T-Lymphocytes, Helper-Inducer

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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