PREreview of BATF controls IFN I production via DC-SCRIPT in plasmacytoid dendritic cells

Major Issues

1. Lack of rescue experiments to establish causality

The mechanistic conclusions rely predominantly on loss-of-function models. Genetic rescue (re-expression of BATF or ZFP366 in knockout cells) or pharmacological rescue targeting the downstream pathway would provide stronger causal evidence that the observed phenotype is directly attributable to disruption of the BATF–ZFP366 axis.

2. Mechanistic validation relies primarily on TLR9 (CpG) stimulation

Most mechanistic experiments were performed using CpG-mediated TLR9 activation. Validation using an additional physiologically relevant stimulus (e.g., TLR7 agonists such as R848 or an independent viral/bacterial model) would determine whether the proposed BATF–DC-SCRIPT pathway represents a general mechanism of pDC activation rather than a TLR9-specific response.

3. Lack of single-cell resolution of IFN-producing cells

The manuscript quantifies overall type I interferon production but does not distinguish whether BATF deficiency increases:

• the proportion of IFN-producing pDCs, or

• the amount of IFN produced per individual cell.

Intracellular IFN staining, IFN reporter mice, or single-cell transcriptomic approaches would substantially strengthen the mechanistic conclusions.

4. Absence of combinatorial genetic (epistasis) analysis

The study investigates BATF, ZFP366, and IFNAR individually but does not examine their combined genetic interactions. Double- and triple-knockout models would clarify whether these molecules function within a single linear pathway or through partially independent mechanisms.

5. Limited characterization of the downstream immune microenvironment

Although BATF deficiency alters IFN-I production, the manuscript does not investigate the downstream immunological consequences. Characterization of immune-cell recruitment (CD4⁺, CD8⁺, B220⁺ B cells, NK cells, myeloid cells) together with chemokine profiling (e.g., CCL5, CXCL9, CXCL10, CCL19/21) would considerably strengthen the biological significance of the findings.

6. No spatial characterization of IFN-producing cells

The study lacks information regarding the anatomical localization of IFN-producing pDCs. Assessing IFN production within the marginal zone, T-cell zone, and B-cell follicles using immunofluorescence, RNAscope, or spatial transcriptomics would provide important physiological context.

7. Insufficient assessment of immune-cell abundance

The manuscript focuses primarily on cytokine production without comprehensively evaluating whether BATF, ZFP366, or IFNAR deficiency alters immune-cell abundance. Quantification of absolute numbers and frequencies of pDCs (B220⁺, CD11c⁺, Siglec-H⁺), together with other immune subsets, would help exclude alterations in cellular composition as a confounding factor.

Minor Issues

1. Validation in conventional dendritic cells (cDCs)

The mechanistic findings are restricted to plasmacytoid dendritic cells. Validation of selected key observations in conventional dendritic cells (cDC1/cDC2) would clarify whether the proposed BATF–DC-SCRIPT regulatory axis is pDC-specific or represents a broader dendritic-cell regulatory mechanism.

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.