Stromal Gasdermin D-mediated Pyroptosis Drives Maladaptive CD4⁺ T-cell Remodeling in Tet2-Deficient Hematopoiesis

Name: Verified Rigor Record — Stromal Gasdermin D-mediated Pyroptosis Drives Maladaptive CD4⁺ T-cell Remodeling in Tet2-Deficient Hematopoiesis
Item: Stromal Gasdermin D-mediated Pyroptosis Drives Maladaptive CD4⁺ T-cell Remodeling in Tet2-Deficient Hematopoiesis
Rating: 75
Author: Αlpha¹

Ji, P.; Ren, K.; Han, X.; Li, E.; Wang, P.; Bi, H.; Cai, W.; Aydemir, I.; Wai, C. M.; Niu, H.; Yang, J.; Liu, Y.; Vadasz, B.; Sukhanova, M.; Fang, D.; Cui, W.

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Αlpha¹ Rigor Review

Rigor Review Verified

Critical

High-priority

Action items to resolve · Critical / High

Confidence: moderateEngine: 7.6.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.

This Alpha1 Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper's main strengths are a well-articulated scientific premise, strong data and code sharing, and transparent reporting of methods, limitations, and funding. However, critical rigor elements are missing: study design lacks described randomization method, blinding, power analysis, and inclusion/exclusion criteria; statistical reporting omits named tests in methods, exact p-values, and effect sizes; ethical approval reporting is incomplete regarding informed consent; and key resource identification (antibodies, software versions) is partial. These gaps reduce reproducibility and should be considered when evaluating the paper's robustness.

The paper combines interventional mouse experiments, observational human sample analysis, and computational bioinformatics. All eight dimensions were applicable and evaluated. Reviewers diverged on study design, biological variables, ethical approvals, key resources, statistical analysis, and reporting transparency; the synthesized judgment weighs the preponderance of checklist evidence. The statistics verification component found no recomputable tests, so no numerical errors were assessed; statistical conclusions should not be treated as independently verified.

12 major claims checked against the paper's own evidence: all adequately supported.

partialResults, 'Patients with isolated TET2 mutations exhibit CD4+ T-cell skewing and tertiary lymphoid structures'Reviewer 2
Patients with isolated TET2 mutations exhibit CD4+ T-cell skewing and CD40L+ T-cell-rich tertiary lymphoid structures in the bone marrow.
The human data are consistent with the claim, but the sample size is not reported, and only qualitative statements are provided; the number of cases and quantitative p-values are absent.
Evidence: Bone marrow samples show increased CD4:CD8 ratio and TLS with CD40L+ T cells; but no N or statistics are given for the human comparison.
“bone marrow samples from patients with isolated TET2 mutations showed a significant increase in the CD4:CD8 T-cell ratio compared with those of normal bone marrow from lymphoma patients without marrow involvement.”
partialResults, 'Stromal pyroptosis licenses the emergence of TFH-like CD4+ T cells'Reviewer 3
Stromal pyroptosis licenses the emergence of TFH-like CD4+ T cells.
The claim is supported by single-cell RNA-seq data showing that TFH-like T cells are expanded in Tet2-/- to wild-type recipients and absent in Gsdmd-/- recipients, but the causal demonstration that stromal pyroptosis alone licenses their emergence (e.g., by inducing pyroptosis in wild-type stroma) is not directly shown; the evidence is correlative.
Evidence: scRNA-seq of bone marrow from Tet2-/- to wild-type recipients showed a marked T-cell population that was dependent on recipient gasdermin D. Subclustering revealed a TFH-like population with Bcl6, Cxcr5, Il21, Cd40l expression that was largely abolished in Gsdmd-/- recipients. CD40L blockade also reduced this population.
“Importantly, the emergence of this TFH-like CD4+ T-cell state was largely abolished in Tet2−/− to Gsdmd−/− recipients, indicating a requirement for stromal gasdermin D-dependent pyroptosis in its induction and maintenance.”
supportedResults, paragraph 2Reviewer 1
Stromal pyroptosis drives Tet2-deficient myeloid expansion.
The claim is supported by transplantation experiments showing that Gsdmd-/- recipients attenuate monocytosis and other disease phenotypes in Tet2-/- to wild-type chimeras.
Evidence: Figure 2 and associated text show that Gsdmd-/- recipients have significant attenuation of monocytosis, anemia, splenomegaly, and histopathological abnormalities.
“Gsdmd −/− recipients showed significant attenuation of monocytosis at 3 months post-transplant”
supportedResults, paragraph 4Reviewer 1
Stromal pyroptosis licenses the emergence of T FH -like CD4 + T cells.
Single-cell RNA sequencing data show that T FH -like cells are expanded in Tet2-/- to wild-type recipients and abolished in Gsdmd-/- recipients.
Evidence: Figure 4 and associated text show that T FH -like cells are present in Tet2-/- to wild-type recipients and absent in Gsdmd-/- recipients.
“the emergence of this T FH -like CD4 + T-cell state was largely abolished in Tet2 −/− to Gsdmd −/− recipients”
supportedResults, paragraph 5Reviewer 1
CD40L blockade breaks the feed-forward loop between T FH -like cells and Tet2-deficient macrophages.
Anti-CD40L treatment reduces monocytosis, splenomegaly, and restores tissue architecture, as shown in Figure 5.
Evidence: Figure 5 shows that anti-CD40L treatment reduces circulating monocytes, splenomegaly, and histopathological abnormalities.
“CD40L blockade resulted in a significant reduction in circulating monocytes compared with IgG-treated controls”
supportedResults, paragraph 6Reviewer 1
Patients with isolated TET2 mutations exhibit CD4 + T-cell skewing and tertiary lymphoid structures.
Human sample analysis shows increased CD4:CD8 ratio and presence of TLS in TET2-mutant patients, but not in DNMT3A-mutant patients.
Evidence: Figure 6 shows increased CD4:CD8 ratio and TLS in TET2-mutant patients.
“bone marrow samples from patients with isolated TET2 mutations showed a significant increase in the CD4:CD8 T-cell ratio”
supportedResults, paragraph 1Reviewer 1
Hematopoietic cell-intrinsic gasdermin D-mediated pyroptosis drives MDS pathogenesis in the mDia1/miR-146a double-knockout model.
The claim is supported by experiments showing that Gsdmd deletion in DKO mice (TKO) rescues MDS phenotypes.
Evidence: Figure 1 shows that TKO mice have ameliorated anemia, thrombocytopenia, and monocytosis.
“Loss of gasdermin D markedly ameliorated MDS phenotypes in DKO mice, including anemia, thrombocytopenia, and monocytosis.”
supportedResults, paragraph 1Reviewer 1
Stromal pyroptosis is secondarily induced by the malignant hematopoietic compartment in the DKO model.
Transplantation of DKO HSPCs into Gsdmd-/- recipients failed to rescue disease, while hematopoietic Gsdmd deletion did, supporting the claim.
Evidence: Figure 1 shows that stromal Gsdmd deficiency fails to rescue DKO phenotypes.
“Stromal gasdermin D deficiency, however, failed to rescue DKO phenotypes or improve survival”
supportedAbstract and ResultsReviewer 2
Stromal pyroptosis drives mutation-specific myeloid expansion by coordinating monocytic remodeling and CD4+ T-cell activation.
The paper provides substantial evidence from transplantation experiments into Gsdmd-/- recipients, spatial transcriptomics, and scRNA-seq showing that stromal pyroptosis is required for Tet2-deficient myeloid expansion and T-cell remodeling.
Evidence: Transplant of Tet2-/- HSPCs into Gsdmd-/- recipients rescues monocytosis and anemia; spatial transcriptomics shows inflammatory macrophage clustering around pyroptotic stromal cells; scRNA-seq shows T FH-like cell emergence dependent on stromal Gsdmd.
“Genetic ablation of gasdermin D in the bone marrow stroma suppressed stromal pyroptosis and attenuated Tet2-deficient myeloid expansion.”
supportedResults, 'CD40L blockade breaks the feed-forward loop'Reviewer 2
CD40L blockade attenuates Tet2-deficient myeloid expansion in vivo.
Anti-CD40L treatment reduced monocytosis, splenomegaly, and tissue infiltration, closely phenocopying the effect of stromal Gsdmd deficiency.
Evidence: Tet2-/- to wild-type chimeras treated with anti-CD40L antibody show significant reduction in circulating monocytes, Ly6C Hi inflammatory monocytes, and normalization of tissue architecture.
“CD40L blockade resulted in a significant reduction in circulating monocytes compared with IgG-treated controls, phenocopying the attenuation observed in Tet2−/− to Gsdmd−/− recipients.”
supportedResults, first two sectionsReviewer 2
Hematopoietic cell-intrinsic pyroptosis drives MDS in the mDia1/miR-146a model, whereas stromal pyroptosis drives Tet2-deficient myeloid expansion.
Both claims are supported by the DKO and Tet2-/- transplantation experiments demonstrating dichotomy.
Evidence: Gsdmd deficiency in donor cells rescues DKO phenotypes, while Gsdmd deficiency in recipients rescues Tet2-/- phenotypes but not DKO phenotypes.
“These results establish that gasdermin D-mediated pyroptosis in hematopoietic cells is the primary cell-autonomous driver of MDS pathogenesis in the DKO model... We therefore investigated whether stromal pyroptosis could serve as the primary driver of TET2-deficient myeloid expansion.”
supportedResults, 'Stromal pyroptosis drives Tet2-deficient myeloid expansion'Reviewer 3
Stromal gasdermin D-mediated pyroptosis drives Tet2-deficient myeloid expansion.
The claim is supported by the key experiment showing that Tet2-/- HSPCs transplanted into Gsdmd-/- recipients have attenuated monocytosis and anemia, rescue of splenomegaly, and reduced histopathology, as presented in the results.
Evidence: Transplantation of Tet2-/- HSPCs into Gsdmd-/- recipients showed significant attenuation of monocytosis at 3 months post-transplant and complete rescue at 9 months, as well as normalization of splenomegaly and histopathology.
“Gsdmd−/− recipients showed significant attenuation of monocytosis at 3 months post-transplant, preceding the development of anemia and thrombocytopenia that manifested at later stages. By 9 months post-transplant, when Tet2−/− to wild-type recipients had developed full disease manifestations, Gsdmd−/− recipients showed complete rescue of both monocytosis and anemia.”

Rigor

Eight Dimensions4 pass · 2 warn · 2 fail

12 major claims2 partial10 supported

16 copyedit issues flagged: mostly clarity, typo, consistency.

Checked 42 references: 37 verified — 5 not checked.

Reproducibility

2 data/code links checked; 2 live.

datahttps://www.10xgenomics.com/support/software/space-ranger/latest/tutorials/count-ffpe-tutorialLIVEHTTP 200
Resolves, but the content could not be matched to the paper.
codeGitHubLIVEHTTP 200
https://github.com/kehanren-sudo/Xenium-Analysis
Resolves to GitHub (code repository).

Alpha¹ Rigor Action ItemsFinal pass before journal submission

The published work is methodologically incomplete in several areas that are critical for reproducibility. An informed reader should weigh the absence of randomization details, blinding, power analysis, and exact statistical reporting when assessing the reliability of the conclusions. These gaps do not necessarily invalidate the findings but warrant cautious interpretation and may justify a correction or independent re-analysis to confirm key claims.

1.
HIGHrigor
Specify the randomization method (e.g., computer-generated random numbers, block allocation) used for assigning animals to experimental groups in the Bone Marrow Transplantation methods via an erratum, or acknowledge the omission for readers.
Without the method, the validity of group allocation cannot be assessed, compromising reproducibility.
2.
HIGHrigor
Describe blinding procedures for investigators performing transplants, outcome assessments, and data analysis, or provide a rationale if blinding was not feasible, in a correction or data note.
Blinding minimizes bias; its absence raises concerns about objectivity of results.
3.
HIGHstatistics
Report exact p-values (e.g., p=0.023) and effect sizes with confidence intervals for all key comparisons in the Results and figure legends, or deposit the underlying data to allow readers to compute them.
Threshold p-values and qualitative 'significant' statements do not allow readers to assess the precision or magnitude of effects.
4.
HIGHstatistics
Name all statistical tests used for each analysis in the Methods section (e.g., 'two-tailed unpaired Student's t-test', 'one-way ANOVA with Tukey post-hoc') rather than only in figure legends.
Central placement in Methods is standard and ensures completeness; current reliance on legends is inconsistent.
5.
HIGHethics
Add a statement on informed consent (or waiver) for the human leftover diagnostic specimens in the Human samples collection methods, as part of an erratum.
The absence of consent information is a reporting gap, even if a waiver was granted by the IRB.
6.
HIGHreporting
Complete the reference to the antibody panels table in the Immunofluorescence methods (currently ends with 'in the .') by providing the actual supplementary table, via a correction.
An incomplete sentence leaves a key resource incompletely documented, hindering replication.
7.
MEDIUMrigor
Report an a priori power analysis or sample size justification for the primary transplantation experiments, or discuss the expected effect sizes and power in a data supplement.
Without power analysis, it is unclear whether the study had sufficient sample size to detect meaningful effects.
8.
MEDIUMrigor
Define pre-specified inclusion/exclusion criteria for mice and human samples, and describe how outliers were identified and handled, in the respective Methods sections.
Lack of these criteria raises uncertainty about potential selective reporting or data exclusion.
9.
MEDIUMreporting
Provide RRIDs for all antibodies and include validation data (e.g., species reactivity, application) in supplementary materials.
RRIDs enable unambiguous identification and facilitate reproducibility across studies.
10.
MEDIUMreporting
Specify the versions of major software tools such as Cell Ranger and Seurat in the Single-cell RNA sequencing methods.
Version numbers are essential for replicating bioinformatic analyses; they are currently omitted for key tools.
11.
MEDIUMreporting
Add a statement that the animal experiments comply with ARRIVE guidelines, or provide a completed checklist in supplementary materials.
ARRIVE guidelines improve reporting of animal research; many journals now expect this reference.
12.
LOWcopyedit
Fix the inconsistent spacing in 'CD4 + T-cell' in the title and ensure consistent capitalization of 'Gsdmd' and 'Tet2-floxed' throughout the manuscript.
Minor inconsistencies detract from professional presentation but do not affect scientific content.

Full text (50,695 chars)Analyzed: Jul 6, 2026

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