Integrity Review: 'IL-35 Stabilizes Treg Phenotype to Protect Cardiac Allografts in Mice' (Huang et al., Transplantation, 2023)

Academic fraud report · Geng Detector

Summary

Verdict: Highly suspicious. This Transplantation paper raises multiple, mutually reinforcing concerns despite plausible reagents and software provenance. The most consequential finding is a methodological implausibility in the in vivo Treg depletion: Foxp3-DTR mice were reportedly dosed with 10 ng/mouse/day diphtheria toxin, an order of magnitude below the 25–50 µg/kg range typically required for effective Treg ablation. If Treg depletion was incomplete, the Figure 4 claim that IL-35 acts through Tregs becomes structurally unsupported. The editorial timeline is also extraordinary: only 3 days elapsed between resubmission (April 14, 2023) and acceptance (April 17, 2023) for a paper involving cardiac transplantation, CyTOF mass cytometry, and mechanistic assays, which is far below normal turnaround for a revised major work. Statistical reporting is internally inconsistent (mean±SEM followed by mean±SD in successive sentences) and Figure 2L reports n=12/group for serum IL-35 versus n=4/group for IFN-γ, TNF-α and IL-2 measured from the same day-7 sera, a 3-fold discrepancy lacking any stated rationale. Pixel-level image analysis was not performed due to lack of raw figure files.

Verdict

🟠 Highly suspicious. Multiple high-severity issues cluster around methodology, editorial handling, and statistical reporting. No image-level duplication could be confirmed or excluded from the available text. None of the concerns are independently fatal, but their combination lowers confidence in the reported findings.

Key findings

Implausibly low diphtheria toxin (DT) dose for Foxp3-DTR Treg depletion (10 ng/mouse/day), versus the standard ~500–1000 ng range; undermines the central mechanistic claim that IL-35 acts via Treg stabilization.
Extraordinary editorial timeline: revision received 2022-12-09, resubmitted 2023-04-14, accepted 2023-04-17 — a 3-day decision window for a complex in vivo + CyTOF manuscript.
Self-contradictory error-bar definition in Methods (mean ± SEM immediately followed by mean ± SD).
Implausible sample-size mismatch in Figure 2L: serum IL-35 reported as n=12/group while IFN-γ, TNF-α, IL-2 from the same day-7 sera reported as n=4/group.
Image-duplication analysis (Western blot splicing, flow cytometry template reuse, IF reuse) was not possible because raw figure files were not accessible.
No temporal anomalies found in software (GraphPad Prism 9.3.1) or reagent catalog numbers.

Evidence highlights

DT dosing (Methods, p.4): "10 ng/mouse/d" via i.p. in Foxp3-DTR mice; standard effective doses in published Foxp3-DTR protocols are ~25–50 µg/kg ≈ 500–1000 ng per 20 g mouse.
Editorial dates: Received 9 Dec 2022; revised 14 Apr 2023; accepted 17 Apr 2023 — 3 days from resubmission to acceptance (DOI: 10.1097/TP.0000000000004707).
Figure 2L legend: IL-35, n=12/group; IFN-γ, TNF-α, IL-2, n=4/group, all stated as day-7 post-transplant sera.
Methods — Statistical Analyses (p.4): consecutive sentences define error bars as "mean ± SEM" and "mean ± SD", without a clear override clause.
Reagent/software: GraphPad Prism 9.3.1 (released late 2021); rIL-35 (AdipoGen, CHI-MF-11135) — temporally consistent with 2022–2023 experiments.

Notes

Confidence: moderate. All flagged issues are textually verifiable, but the absence of image files means Western blot splicing or flow-plot reuse cannot be ruled in or out.
Recommended follow-ups: (1) request raw FCS files for Figure 4 Treg depletion panels to verify DT efficacy; (2) request original lab-book DT dosing records; (3) request clarification on the n=12 vs n=4 discrepancy in Figure 2L and on the SEM/SD contradiction; (4) consider formal inquiry to *Transplantation* regarding the 3-day resubmission-to-acceptance interval and reviewer identities.