Metabolically Activated Proteostasis Regulators Reduce Differentiation of CD4+ TH17 Cells
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eLife Assessment
This study provides valuable evidence that the metabolically activated proteostasis regulator AA147 preferentially suppresses T helper 17 cell differentiation, implicating nuclear factor erythroid 2-related factor 2 (NRF2), reduced reactive oxygen species (ROS) levels, and proteasome-dependent degradation of RORγt in this effect. The findings are supported by solid evidence from complementary pharmacological, genetic, biochemical, transcriptomic, proteomic, and cellular approaches. However, the precise mechanistic link between NRF2 activation, ROS reduction, and RORγt degradation remains to be fully elucidated.
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Abstract
The differentiation of naïve CD4+ T cells into effector T cell subsets such as TH1, TH2, TH17 and TREG cells is governed by tightly coordinated activation programmes dependent on T cell receptor (TCR) engagement, co-stimulation, and cytokine signalling. This differentiation process involves regulation of stress-responsive signalling pathways such as the unfolded protein response (UPR) and the oxidative stress response (OSR) to adapt to the physiologic demands specific to distinct T cell subset functions. This suggests that pharmacologically targeting stress-responsive pathways offers a unique opportunity to selectively remodel the differentiation of these different T cell subsets. We previously identified compound AA147 as a metabolically activated proteostasis regulator that can induce both the ATF6 signalling arm of the UPR and, in certain cell types, the NRF2-regulated arm of the OSR (Paxman et al., 2018; Plate et al., 2016). Here, we show that treatment with AA147 selectively reduces differentiation of pro-inflammatory TH17 cells by promoting degradation of the lineage-specifying transcription factor RORγt, without impacting the transcription factors of other CD4+ T cell subsets. We find that AA147-dependent reduction in TH17 differentiation is independent of ATF6 activation but involves NRF2 activation and reductions in intracellular reactive oxygen species (ROS). Apart from RORγt, we further show that AA147 decreases expression of the TCR-responsive factor IRF4, thus suppressing production of select effector cytokines across effector T cells, revealing additional ways in which this compound reshapes the activities of these essential T cell subsets. Our results demonstrate the potential for metabolically activated proteostasis regulators such as AA147 to selectively reshape TH17 cell identity while broadly dampening effector cytokine responses across effector T cell subsets.
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eLife Assessment
This study provides valuable evidence that the metabolically activated proteostasis regulator AA147 preferentially suppresses T helper 17 cell differentiation, implicating nuclear factor erythroid 2-related factor 2 (NRF2), reduced reactive oxygen species (ROS) levels, and proteasome-dependent degradation of RORγt in this effect. The findings are supported by solid evidence from complementary pharmacological, genetic, biochemical, transcriptomic, proteomic, and cellular approaches. However, the precise mechanistic link between NRF2 activation, ROS reduction, and RORγt degradation remains to be fully elucidated.
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Reviewer #1 (Public review):
Summary:
Beginning with the rationale that the unfolded protein response (UPR) and oxidative stress response (OSR) both impact T cell differentiation and effector functions, the authors investigated whether a metabolically activated drug (AA147) shown to enhance the UPR and (via electrophilic disinhibition of Nrf2) OSR can alter CD4 fate and function. Using pharmacologic and focused genetic manipulations in cultured mouse CD4 cells, they found that AA147 attenuated Th17 cell differentiation via Nrf2 activation. In other CD4 subsets, they found that AA147 had no effect on differentiation but nonetheless decreased effector cytokine expression via an independent effect on IRF4. Based on these results, the authors propose that AA147 and similar drugs might be candidate therapies for autoimmune diseases, many of …
Reviewer #1 (Public review):
Summary:
Beginning with the rationale that the unfolded protein response (UPR) and oxidative stress response (OSR) both impact T cell differentiation and effector functions, the authors investigated whether a metabolically activated drug (AA147) shown to enhance the UPR and (via electrophilic disinhibition of Nrf2) OSR can alter CD4 fate and function. Using pharmacologic and focused genetic manipulations in cultured mouse CD4 cells, they found that AA147 attenuated Th17 cell differentiation via Nrf2 activation. In other CD4 subsets, they found that AA147 had no effect on differentiation but nonetheless decreased effector cytokine expression via an independent effect on IRF4. Based on these results, the authors propose that AA147 and similar drugs might be candidate therapies for autoimmune diseases, many of which are associated with pathologic Th17 cells.
Strengths:
The experimental design and associated data are generally appropriate and sufficient to support the authors' conclusions. Although several conclusions rest on pharmacologic rather than genetic interventions, they utilized genetic manipulation in key studies to support their conclusions. Namely, they utilized Nrf2 KO cells to demonstrate the necessity of Nrf2 for AA147 effects on Th17 differentiation.
Weaknesses:
A general weakness is the lack of in vivo data to support their findings. From the literature, it appears that AA147 can be given to intact mice with beneficial effects in autoimmune models such as experimental autoimmune encephalomyelitis (EAE). This would provide the opportunity to confirm the decreased Th17 differentiation and the proposed mechanism in a relevant animal model.
Because of the reliance on pharmacologic interventions, some of the conclusions are also based on somewhat indirect evidence that assumes both the specificity and completeness of on-target effects.
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Reviewer #2 (Public review):
Summary:
This manuscript investigates how the metabolically activated proteostasis regulator AA147 influences CD4+ T-cell differentiation. The authors provide evidence that AA147 preferentially suppresses TH17 differentiation, reduces RORγt protein abundance without substantially decreasing Rorc transcription, and engages an NRF2-dependent antioxidant response associated with reduced intracellular ROS. In addition, the study identifies a broader, NRF2- and ATF6-independent reduction in IRF4 that is accompanied by decreased effector cytokine production across several CD4+ T-cell subsets.
Strengths:
The work addresses an interesting intersection between proteostasis, redox regulation, and T-cell differentiation, and several aspects of the study are compelling. In particular, the combination of genetic NRF2 …
Reviewer #2 (Public review):
Summary:
This manuscript investigates how the metabolically activated proteostasis regulator AA147 influences CD4+ T-cell differentiation. The authors provide evidence that AA147 preferentially suppresses TH17 differentiation, reduces RORγt protein abundance without substantially decreasing Rorc transcription, and engages an NRF2-dependent antioxidant response associated with reduced intracellular ROS. In addition, the study identifies a broader, NRF2- and ATF6-independent reduction in IRF4 that is accompanied by decreased effector cytokine production across several CD4+ T-cell subsets.
Strengths:
The work addresses an interesting intersection between proteostasis, redox regulation, and T-cell differentiation, and several aspects of the study are compelling. In particular, the combination of genetic NRF2 deficiency, pharmacological pathway perturbation, RNA-seq, quantitative proteomics, and testing under distinct TH17-polarizing conditions provides multiple complementary lines of evidence. The observation that AA147 reduces RORγt without substantially altering Rorc transcription also raises an interesting possibility of post-translational control of TH17 lineage specification.
Weaknesses:
Several of the central mechanistic conclusions currently extend beyond what is directly demonstrated. In particular, the proposed causal pathway linking AA147-induced NRF2 activation and reduced ROS to proteasomal degradation of RORγt remains incompletely established. Moreover, the broad effects of AA147 on IRF4 and cytokine production across multiple CD4+ T-cell subsets complicate the claim of selective TH17 targeting. Finally, the current study is largely restricted to ex vivo murine differentiation, whereas the Discussion emphasizes potential therapeutic application in inflammatory disease. Addressing the points below would substantially strengthen both the mechanistic depth and the overall interpretation of the study.
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Reviewer #3 (Public review):
Summary:
The authors investigate how AA147, a metabolically activated proteostasis regulator, affects CD4-positive T-cell differentiation. The study shows that AA147 preferentially suppresses ex vivo TH17 differentiation and reduces RORγt protein through a proteasome-dependent mechanism. The authors further provide evidence that nuclear factor erythroid 2-related factor 2 and reduced reactive oxygen species contribute to this phenotype, while demonstrating that AA147 also reduces IRF4 across multiple CD4-positive T-cell subsets through a mechanism that is independent of nuclear factor erythroid 2-related factor 2 and ATF6. The study combines pharmacological perturbation, genetic analysis, protein stability experiments, transcriptomics, proteomics, and analysis of multiple T-cell subsets. Overall, the data …
Reviewer #3 (Public review):
Summary:
The authors investigate how AA147, a metabolically activated proteostasis regulator, affects CD4-positive T-cell differentiation. The study shows that AA147 preferentially suppresses ex vivo TH17 differentiation and reduces RORγt protein through a proteasome-dependent mechanism. The authors further provide evidence that nuclear factor erythroid 2-related factor 2 and reduced reactive oxygen species contribute to this phenotype, while demonstrating that AA147 also reduces IRF4 across multiple CD4-positive T-cell subsets through a mechanism that is independent of nuclear factor erythroid 2-related factor 2 and ATF6. The study combines pharmacological perturbation, genetic analysis, protein stability experiments, transcriptomics, proteomics, and analysis of multiple T-cell subsets. Overall, the data support the central conclusion that AA147 preferentially affects TH17 differentiation and alters RORγt protein stability, although the molecular connection between nuclear factor erythroid 2-related factor 2, reactive oxygen species, and RORγt degradation remains incompletely defined.
Strengths:
A major strength is the use of complementary experimental approaches to establish the effect of AA147 on TH17 differentiation. The authors show reduced RORγt-positive and IL-17A-positive cells while preserving the examined lineage-defining transcription factors in TH1, TH2, and TREG cells, and they reproduce the phenotype under different TH17-polarizing conditions. The RORγt stability experiments are particularly strong: AA147 does not reduce Rorc transcription, and proteasome inhibition with bortezomib prevents the AA147-dependent reduction in RORγt protein, supporting a post-translational mechanism. The distinction between ATF6 and NRF2 signaling is also supported by complementary pharmacological and genetic experiments, including NRF2-deficient CD4-positive T cells and an independent NRF2 activator. The study is further strengthened by RNA sequencing and quantitative proteomics and by examination of AA147 effects across several CD4-positive T-cell subsets. The authors also provide biological replicates for the major experiments and make the sequencing and proteomic datasets available.
Weaknesses:
The main limitation is that the molecular pathway connecting NRF2 activation and reduced reactive oxygen species to proteasome-dependent RORγt degradation is not fully established. The experiments support a model in which altered redox state contributes to RORγt loss, but they do not directly define how reduced reactive oxygen species promotes RORγt proteasomal degradation. In addition, although the study demonstrates that AA147 reduces IRF4 independently of NRF2 and ATF6 across multiple CD4-positive T-cell subsets, the mechanism responsible for this effect remains unresolved. Finally, the proteomic analysis should clarify what constituted the pairing of the three vehicle-treated and three AA147-treated samples, since the Methods state that paired statistical testing was used. These limitations do not undermine the central findings but should be clearly distinguished from the mechanistic conclusions that are directly supported by the data.
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Metabolically Activated Proteostasis Regulators Reduce Differentiation of CD4+ TH17 Cells https://www.biorxiv.org/content/10.64898/2026.09.02.747634v1
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Metabolically Activated Proteostasis Regulators Reduce Differentiation of CD4+ TH17 Cells https://www.biorxiv.org/content/10.64898/2026.09.02.747634v1
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