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Major Issues

1. PBMC model & Organ-on-chip model

Although these in vitro models provide valuable evidence that IL-38 suppresses alloreactive immune responses, several mechanistic analyses are missing that would strengthen the authors' conclusions.

• IL-17 was not included in the cytokine profile. Given the well-established association between IL-38 deficiency and enhanced Th17/IL-17 responses, measuring IL-17 alongside the other inflammatory cytokines would help determine whether IL-38 also regulates the Th17 axis in these experimental systems.

• CD39 expression was not evaluated on FoxP3⁺ Tregs. While the authors demonstrated an increase in the frequency of FoxP3⁺ regulatory T cells, they did not investigate whether these cells also acquire an enhanced suppressive phenotype. Assessing the percentage or MFI of CD39 on FoxP3⁺ Tregs would provide mechanistic evidence supporting activation of the CD39/CD73 immunosuppressive pathway.

• Functional characterization of Tregs remains incomplete. The increase in FoxP3⁺ cells demonstrates numerical expansion but does not necessarily indicate enhanced suppressive capacity. Additional phenotypic markers, particularly CD39, would strengthen the conclusion that IL-38 promotes functional regulatory T cells rather than simply increasing their frequency.

2. GVHD model (Blood & Spleen)

The in vivo blood and spleen experiments primarily focused on immune cell frequencies but lacked a comprehensive assessment of the inflammatory cytokine milieu.

• Since these samples are not suitable for tissue infiltration analyses in the same manner as liver or intestinal tissues, a comprehensive cytokine profile would provide a more informative assessment of systemic immune regulation.

• IL-17 should be included among the analyzed cytokines. Because IL-17 is closely linked to IL-38 biology and GVHD-associated inflammation, its measurement would substantially improve the mechanistic interpretation of the observed protective effects.

• Measuring additional inflammatory cytokines together with IL-17 would also help correlate systemic cytokine modulation with the observed changes in T-cell activation and regulatory T-cell expansion.

3. GVHD model (Liver & Intestine)

Although the tissue analyses demonstrated reduced immune cell infiltration following IL-38 treatment, several important cellular populations and mechanistic experiments were omitted.

• Neutrophil infiltration was not evaluated. Neutrophils are major mediators of tissue injury during acute GVHD, particularly within the liver and gastrointestinal tract. Quantification of Ly6G⁺ neutrophils by flow cytometry or immunohistochemistry would strengthen the pathological interpretation of IL-38-mediated protection.

• γδ T-cell infiltration was not assessed. Since γδ T cells can rapidly produce IL-17 and contribute significantly to tissue inflammation, their quantification would provide additional mechanistic insight into how IL-38 regulates inflammatory responses within GVHD target organs.

• The CD39 pathway was not functionally validated. A mechanistic experiment using the CD39 inhibitor POM-1, followed by quantification of neutrophil infiltration and disease severity, would determine whether the protective effects of IL-38 are dependent on CD39-mediated immunosuppression. This experiment would directly connect IL-38 signaling, regulatory T-cell function, and tissue inflammation.

Minor Issues

1. Cellular source of IL-38

The study does not identify the principal cellular source of IL-38 responsible for the observed protective effects. Cell type-specific IL-38 deletion models (e.g., epithelial cells, macrophages, or B cells) would help determine which cellular compartment contributes most significantly to immune regulation during GVHD and would further strengthen the mechanistic conclusions.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they used generative AI to come up with new ideas for their review.