Dear Dr. Mohamed,

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Francesco Bertolini, MD, PhD

Academic Editor

PLOS ONE

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“OG: Assar Gabrielsson foundation, Anna-Lisa and Bror Björnsson foundation, Adlerbert Research Foundation, the Royal Sciences and Arts in Gothenburg and the

Austrian Science Fund (FWF), project PAT3959823.

MT: Associazione Italiana per la Ricerca sul Cancro (IG28768).

AM: European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie agreement No101034307”

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This study offers a novel investigation into CLL subtype origins using germinal center trajectory mapping. The concept is compelling and well-executed, but methodological caveats require clarification to solidify conclusions.

The application of tonsillar GC B-cell markers (Holmes et al.) to infer developmental states in peripheral blood-derived CLL cells presents biological disconnects:

• Tonsillar GC substages rely on niche-specific signals (e.g., CXCL12/CD40L) absent in circulation, risking misinterpretation of transcriptomic signatures as active GC maturation.

• Protein-level validation (e.g., CXCR4/CD83 flow cytometry) and comparison to lymph node/bone marrow microenvironments are lacking.

Could the observed substage signatures reflect aberrant transcriptional mimicry (e.g., constitutive NF-κB) rather than true GC maturation? Please temper claims and discuss limitations.

Additionally, please Revise Methods to passive voice for objectivity.

Reviewer #2: In the manuscript, the authors have tried

1) To investigate the potential cellular origin of M- and UM-CLL subtypes using multiple CLL transcriptomic cohorts downloaded from public databases and compared the data RNA sequencing data generated in their lab from mature-naïve (CD24+CD38int) B cells, CD27dull MBCs (CD24hiCD27dull) and CD27bright MBCs (CD24hiCD27bright).

2) To decipher commonalities and differences between M- and UM-CLL subtypes against healthy B cells through a transcriptomic meta-analysis and,

3) To investigate the potential GC origin of the two CLL subtypes using single-cell data already available.

Specific Comments:

The manuscript title 'Germinal center trajectories and transcriptional signatures define CLL subtypes and their functional regulators,' suggests the inclusion of functional assays. However, based on the manuscript's content, the proposed pathways and gene signature relationships appear to be derived solely from in silico pathway analysis and the available literature. To accurately reflect the scope of the work, it is recommended to revise the title particularly regarding the 'functional regulators' aspect.

The same implies in the abstract also. The authors have mentioned that functional analysis revealed that LPL…..EMT regulation. The term functional analysis should be replaced with functional enrichment analysis.

The abstract needs to clearly outline the methodology used in the manuscript. It needs to include details like number of patients studied, methodology like meta analysis was conducted.

The transcriptional signatures highlighting the traditional and potential biomarkers for CLL sub- types is diverting the focus of the manuscript. Many published papers are already available for transcriptional signatures between these subtypes (Mutated vs unmutated). Hence, the abstract and discussion should be modified accordingly.

Given that the results are based on bioinformatics meta-analysis without functional validation, avoid making very strong conclusions. Please use mild words like 'suggested' or 'hypothesized' in both the abstract and discussion. A statement acknowledging that these findings require functional validation may be added somewhere in the discussion.

The effort should be to make it concise. In the introduction section, paragraph 2 may be more concise. The discussion also needs to be modified and concise in line with the suggestions provided above.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes:  Amir Abbas Navidinia

Reviewer #2: Yes:  Ritu Gupta

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Germinal center trajectories and transcriptional signatures define CLL subtypes and their pathway regulators

PONE-D-25-22015R1

Dear Dr. Mohamed,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Francesco Bertolini, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes:  Amir Abbas Navidinia

Reviewer #2: Yes:  Ritu Gupta

**********