First Comprehensive Analysis of Full-Length and Δ2 Foxp3 Isoforms Distribution in PBMCs from Healthy Volunteers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled *"FOXP3 Isoform Distribution and PD-1/CTLA-4 Expression in Peripheral Blood Immune Cells from Healthy Donors"* presents a well-executed descriptive flow cytometry study addressing an underexplored area of FOXP3 biology. The data are novel, clearly presented, and support the main conclusions. However, several minor issues—including typographical errors, figure labeling inconsistencies, and areas requiring clarification in Methods and Discussion—should be addressed to improve clarity and reproducibility. The following minor revision comments are offered to strengthen the manuscript for publication in Biomolecules.

1. Abstract & Introduction

• Line 15–17: The statement *“FOXP3-Δ2 was more frequently detected in CD8+ T cells, monocytes, and neutrophils”* is clear, but consider adding a brief note that absolute frequencies in these populations are very low (as shown in Results) to avoid overinterpretation.

• Line 43–46: The introduction of FOXP3 splicing is well written, but the transition from general Treg biology to isoform-specific roles could be tightened by moving the mention of murine studies (lines 59–62) to a later point in the paragraph.

2. Materials and Methods

• Line 95: Specify the concentration or volume of Fixable Viability Stain 510 used, or state that it followed the manufacturer’s recommended titration.

• Line 104: Clarify whether the second lysis step (if necessary) used the same buffer or a different protocol.

• Line 117: “Figure X” is a placeholder – replace with the correct figure number.

3. Results

• Line 140: The statement “No data on donor age or sex were collected” is appropriate, but consider mentioning in the Discussion whether this limits generalizability (e.g., known sex differences in FOXP3 expression).

• Figure 2B: Absolute event counts are informative, but the y-axis label should explicitly state “per 500 μL peripheral blood” (already in legend – move to axis label).

• Table 1: The layout is difficult to parse. Recommend splitting into two tables or using clearer column headers (e.g., separate % and Events for each isoform combination).

• Line 190: “FOXP3-FL+Δ2+” appears twice with different meanings; revise for clarity (likely typo: one should be FL+Δ2- or similar).

• Figure 4, 6, 8, 10, 12: The use of multiple symbols (*, **, ***, #) is fine, but ensure each symbol is explicitly defined in every legend (some figures omit the definition of #).

4. Discussion

• Line 515–517: The statement that FOXP3-Δ2 predominance in non-CD4+ populations “suggests that isoform distribution is associated with cell lineage” is reasonable, but acknowledge that protein detection sensitivity may also play a role (low overall expression).

• Line 543–545: The phrase “to our knowledge… has not been systematically explored” is fine, but consider citing any prior proteomic or single-cell RNA-seq studies that might have noted FOXP3 transcripts in myeloid cells.

• Line 580–582: The suggestion that higher PD-1 in FOXP3-Δ2+ cells “may reflect a compensatory mechanism” is speculative – soften to “could be consistent with” or “hypothesized to represent”.

• Line 30: “Pharaocogenomics” → should be “Pharmacogenomics”.

• Line 89: Remove extra space before “90”.

• Line 227: “FOXP” → should be “FOXP3”.

• Line 459: “Table 10” is referenced but not fully shown in the provided PDF – verify completeness.

• Line 629: AI-assisted tools disclosure is commendable – add a brief note that no AI was used for data interpretation or final conclusions.

Author Response

The manuscript titled *"FOXP3 Isoform Distribution and PD-1/CTLA-4 Expression in Peripheral Blood Immune Cells from Healthy Donors"* presents a well-executed descriptive flow cytometry study addressing an underexplored area of FOXP3 biology. The data are novel, clearly presented, and support the main conclusions. However, several minor issues—including typographical errors, figure labeling inconsistencies, and areas requiring clarification in Methods and Discussion—should be addressed to improve clarity and reproducibility. The following minor revision comments are offered to strengthen the manuscript for publication in Biomolecules.

First of all, we would like to thank the reviewer for the detailed review of the manuscript and the valuable comments that we are confident that will improve the overall quality of our work.

1. Abstract & Introduction

• Line 15–17:The statement *“FOXP3-Δ2 was more frequently detected in CD8+ T cells, monocytes, and neutrophils”* is clear, but consider adding a brief note that absolute frequencies in these populations are very low (as shown in Results) to avoid overinterpretation.

Thanks for the suggestion. We have included the following sentence to avoid misunderstanding: ‘whose absolute frequencies are low though’

 

• Line 43–46:The introduction of FOXP3 splicing is well written, but the transition from general Treg biology to isoform-specific roles could be tightened by moving the mention of murine studies (lines 59–62) to a later point in the paragraph.

Thanks for the suggestion, the mention of murine studies has been moved forward accordingly.

 

2. Materials and Methods

• Line 95:Specify the concentration or volume of Fixable Viability Stain 510 used, or state that it followed the manufacturer’s recommended titration.

To clarify this point, we have added the following sentence: ‘following the manufacturer’s recommendation’

• Line 104:Clarify whether the second lysis step (if necessary) used the same buffer or a different protocol.

The following sentence has been included to make this point clearer: ‘using the same lysis buffer’

• Line 117:“Figure X” is a placeholder – replace with the correct figure number.

Sorry for the mistake. This has been corrected

 

3. Results

• Line 140:The statement “No data on donor age or sex were collected” is appropriate, but consider mentioning in the Discussion whether this limits generalizability (e.g., known sex differences in FOXP3 expression).

Thanks for the suggestion. We have included the following sentence within the discussion to clarify this: ‘Not considering the sex of the patients as a parameter could limit the generalizability of the results, although its effect on FOXP3 expression remains controversial, as different studies have reported conflicting findings [39,40].’

• Figure 2B:Absolute event counts are informative, but the y-axis label should explicitly state “per 500 μL peripheral blood” (already in legend – move to axis label).

Thanks for the suggestion. We have modified it within the new version of the manuscript by adding the following sentence in the y axis label: ‘ / 500 μL peripheral blood’

• Table 1:The layout is difficult to parse. Recommend splitting into two tables or using clearer column headers (e.g., separate % and Events for each isoform combination).

Thanks for your observation. We have now included a double line to separate the % and event columns more clearly

• Line 190:“FOXP3-FL+Δ2+” appears twice with different meanings; revise for clarity (likely typo: one should be FL+Δ2- or similar).

Sorry for the mistake, it has been corrected in the new version of the manuscript

• Figure 4, 6, 8, 10, 12:The use of multiple symbols (*, **, ***, #) is fine, but ensure each symbol is explicitly defined in every legend (some figures omit the definition of #).

The reviewer is right. The figure legends have been modified accordingly

 

4. Discussion

• Line 515–517:The statement that FOXP3-Δ2 predominance in non-CD4+ populations “suggests that isoform distribution is associated with cell lineage” is reasonable, but acknowledge that protein detection sensitivity may also play a role (low overall expression).

Thanks for underlining this fact. We have added the following sentence to clarify this point: ‘However, it must be taken into account that the technical detection sensitivity could play a role in the low overall expression detected.’

• Line 543–545:The phrase “to our knowledge… has not been systematically explored” is fine, but consider citing any prior proteomic or single-cell RNA-seq studies that might have noted FOXP3 transcripts in myeloid cells.

Thanks for the comment, we have added the following references:

[34] Devaud C, Darcy PK, Kershaw MH. Foxp3 expression in T regulatory cells and other cell lineages. Cancer Immunol Immunother. 2014 Sep;63(9):869-76. doi: 10.1007/s00262-014-1581-4.

[35] Ilnicka A, Gocek E, Łopatecka J, Marcinkowska E. Regulation of FOXP3 expression in myeloid cells in response to all-trans-retinoic acid, interleukin 2 and transforming growth factor β. Dev Comp Immunol. 2019 Jul;96:18-26. doi: 10.1016/j.dci.2019.02.019.

• Line 580–582:The suggestion that higher PD-1 in FOXP3-Δ2+ cells “may reflect a compensatory mechanism” is speculative – soften to “could be consistent with” or “hypothesized to represent”.

We totally agree with the reviewer. We have modified it by including the suggested expression ‘could be consistent with’

• Line 30:“Pharaocogenomics” → should be “Pharmacogenomics”.

Sorry for the mistake, it has been corrected

• Line 89:Remove extra space before “90”.

It has been removed

• Line 227:“FOXP” → should be “FOXP3”.

It has been corrected

• Line 459:“Table 10” is referenced but not fully shown in the provided PDF – verify completeness.

We do not know if there was a mistake. We can see it in page 19 of the manuscript downloaded from the revision page.

• Line 629:AI-assisted tools disclosure is commendable – add a brief note that no AI was used for data interpretation or final conclusions.

Thanks for the suggestion. The sentence proposed ‘no AI was used for data interpretation or final conclusions’ has been added.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors

 

Hi. In advance, I appreciate your attention to this message. So, while I consider that the study is interesting, and more if it is dealing with regulatory T cells, teh implication of them in keeping in tone and in homeostasis of the immune response, either by innate and adaptive cells. It is necessary and utmost important to include in the research design at least some individuals with different affections. Otherwise, it's good to have the data, but nowadays we already need translational medicine addressing different infectious and non infectious. I found that the comparison with the checkpoints would be great to start from the introduction, and really to analyze the relationship, in addition to theoretically, with individuals with some type of cancer. A scheme on this relationship is also highly recommendable. 

Comments for author File: Comments.pdf

Author Response

Dear Authors

 Hi. In advance, I appreciate your attention to this message. So, while I consider that the study is interesting, and more if it is dealing with regulatory T cells, the implication of them in keeping in tone and in homeostasis of the immune response, either by innate and adaptive cells. It is necessary and utmost important to include in the research design at least some individuals with different affections. Otherwise, it's good to have the data, but nowadays we already need translational medicine addressing different infectious and non infectious. I found that the comparison with the checkpoints would be great to start from the introduction, and really to analyze the relationship, in addition to theoretically, with individuals with some type of cancer. A scheme on this relationship is also highly recommendable. 

We truly appreciate the detailed review of the referee. We believe that the changes implemented in response to the reviewer's suggestions have significantly strengthened the manuscript and improved its overall quality

ANSWERS

We thank the reviewer for this valuable suggestion. We agree that the biological and clinical relevance of FOXP3 isoforms extends beyond their characterization in healthy individuals and deserves discussion in the context of immune-mediated diseases. We agree that studying FOXP3 isoforms in pathological conditions would provide valuable translational information. However, the objective of the present work was to establish a comprehensive baseline characterization of FOXP3 isoform expression across immune cell subsets in healthy individuals and a cytometry protocol to evaluate this simultaneously. We have now acknowledged this limitation in the Discussion and highlighted future studies in patients with cancer, autoimmune diseases, and infectious diseases as an important next step by including the following sentence: ‘Studying FOXP3 isoforms in pathological conditions would provide valuable transla-tional information. However, the objective of the present work was to establish a com-prehensive baseline characterization of FOXP3 isoform expression across immune cell subsets in healthy individuals and a cytometry protocol to evaluate this simultaneously’.

We have also incorporated new paragraphs within the introduction and discussion sections discussing the relationship between FOXP3 isoforms, regulatory T-cell biology, and immune checkpoint pathways, including CTLA-4, PD-1, TIGIT, and LAG-3. We also discuss the potential relevance of differential FOXP3 isoform expression in cancer, autoimmune diseases, and chronic inflammatory conditions, where altered regulatory T-cell activity contributes to disease pathogenesis and therapeutic responses.

 

New paragraphs included in different parts of the manuscript:

Introduction

‘It has been proved that the expression of FOXP3-Δ2 is associated with promotion of CD8 T cell–mediated antitumor immunity, this providing resistance to the develop-ment of multiple types of tumors [16]. Increased or imbalanced expression of ΔE2 in relation to FL is often linked to Treg dysfunction and autoimmunity.’

‘Regulatory T cells exert suppressive functions through multiple mechanisms, including the expression of immune checkpoint molecules such as CTLA-4, PD-1, TIGIT, and LAG-3, many of which are directly or indirectly regulated by FOXP3 [17,18]. ‘

…

‘Given the increasing clinical relevance of immune checkpoint modulation in cancer, autoimmunity, and chronic inflammatory diseases, understanding the distribution of FOXP3 isoforms across immune cell populations may provide valuable insights into mechanisms of immune regulation and potential biomarkers of immune dysfunction.’

Discussion

‘Beyond the descriptive characterization presented here, our findings may have important implications for understanding immune regulation in pathological condi-tions. Regulatory T cells are highly enriched in many tumors and constitute a major mechanism of immune suppression within the tumor microenvironment. Their suppressive activity is closely associated with the expression of immune checkpoint mol-ecules, including CTLA-4, PD-1, TIGIT, and LAG-3, which are currently targeted by several immunotherapeutic strategies.’

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript "Distribution of full-length and Δ2 variants of Foxp3 in PBMCs of healthy volunteers", written by Fernandez-Delgrado M, Sendra L, Herrero MJ, Olivera-Pasquini  GG, Zucchet EG, Garcia-Boyero R and Alino SF, analyses expression of FOXP3, full-length and exon 2 deleted variant, in different types of leukocytes, and the relations to expression of immune checkpoints. As the role of FOXP3 is known in regulatory T cells, the authors analyze also other types of leukocytes.

The manuscript is well written with appropriate description of methods and results. However, the main comments are the same as the authors put at the end of discussion: what is the biological significance of FOXP3 expression in non-CD4 population. In some populations the level of FOXP3 expression is very low, so there is a question whether complex statistical analysis should be done.

In the Introduction, more data on the FOXP3 regulation can be given, whether it controls expression of immune checkpoint proteins directly (such as CTLA4?), how is it deregulated in diseases, what happened when  CD4CD25- cells were transduced with FOXP3.

In the Results, an introductory sentence can explain what the samples were and what was done with them, and why.

One histogram with all cell types presenting FOXP3 expression (in percentage), for both, total FOXP3 expression and both isoforms can be added.

For Discussion:

it would be more informative if comparison of percentage of FOXP3 + cells among Treg CD4CD25 in other published results from control samples and explanation of their relatively low percentage is added; in the discussion it is not accented that CD4CD25 belong to Treg;

The biological significance of FOXP3 expression in non-CD4 population and hypotheses of the possible consequences of their expression can be discussed;  the role of FOXP3 and its variants in Treg development; role of cell signaling network in the outcomes of FOXP3 expression, functions of deleted form of FOXP3, possible mechanisms of their translation regulation.

Can data from RNAseq from different types of leukocytes be extracted and compared with obtained data?

 

Other comments

signs + and – can be bigger in the figures.

In figure legends a number of samples can be given

line  336 vent ratio?

line 447: sentence reorganization

Author Response

The manuscript "Distribution of full-length and Δ2 variants of Foxp3 in PBMCs of healthy volunteers", written by Fernandez-Delgrado M, Sendra L, Herrero MJ, Olivera-Pasquini  GG, Zucchet EG, Garcia-Boyero R and Alino SF, analyses expression of FOXP3, full-length and exon 2 deleted variant, in different types of leukocytes, and the relations to expression of immune checkpoints. As the role of FOXP3 is known in regulatory T cells, the authors analyze also other types of leukocytes. The manuscript is well written with appropriate description of methods and results. However, the main comments are the same as the authors put at the end of discussion: what is the biological significance of FOXP3 expression in non-CD4 population. In some populations the level of FOXP3 expression is very low, so there is a question whether complex statistical analysis should be done.

We thank the reviewer for the careful evaluation of our manuscript and for the constructive suggestions. We agree that the biological significance of FOXP3 expression in non-CD4+ leukocyte populations represents one of the most important questions arising from our findings. In response, we have expanded the Introduction and Discussion sections to provide additional information regarding FOXP3 regulation, its relationship with immune checkpoint molecules, its role in regulatory T-cell biology, and current evidence supporting FOXP3 expression in non-conventional immune cell populations.

Regarding the statistical analyses, we acknowledge that FOXP3 expression levels in some leukocyte populations were substantially lower than those observed in the classical CD4+CD25+ regulatory T-cell compartment. However, FOXP3 expression remained consistently detectable across biological replicates and above the analytical detection threshold of the assay. To address the reviewer's concern, we have expanded the Discussion to emphasize that ‘statistically significant differences detected in populations exhibiting low FOXP3 expression should be interpreted cautiously. While these findings may indicate the existence of low-level regulatory programs outside the conventional Treg compartment, additional functional studies will be required to establish their biological relevance’.

In the Introduction, more data on the FOXP3 regulation can be given, whether it controls expression of immune checkpoint proteins directly (such as CTLA4?), how is it deregulated in diseases, what happened when CD4CD25- cells were transduced with FOXP3.

We thank the reviewer for this valuable suggestion. We have expanded the Introduction to provide additional background on FOXP3 biology. Specifically, we now describe its role as a master transcriptional regulator of regulatory T-cell function, including its regulation of immune checkpoint molecules such as CTLA-4. We also added information regarding mechanisms leading to FOXP3 dysregulation in autoimmune diseases, inflammatory disorders, and cancer. Finally, we included a paragraph within the introduction on how ectopic expression of FOXP3 in conventional CD4+CD25− T cells induces a regulatory-like phenotype characterized by suppressive activity and altered expression of Treg-associated genes by including the following sentence: ‘Experimental studies have further demonstrated that ectopic expression of FOXP3 in conventional CD4+CD25− T cells can confer suppressive properties and induce the expression of several Treg-associated markers (GITR, CTLA-4, and TGFβ), suppress the proliferation of naïve CD4+T cells and inhibit the activation-induced IL-2 gene expression and the production of IFNϒ in response to TCR ligation [19], supporting its central role in establishing the regulatory T-cell phenotype’.

In the Results, an introductory sentence can explain what the samples were and what was done with them, and why.

To improve data presentation, we have added an introductory paragraph to the Results section describing the analyzed samples and the rationale for the experimental design: ‘Peripheral blood samples from six healthy donors were analyzed by multiparametric flow cytometry to describe the basal frequency and distribution of the FOXP3-FL and FOXP3-Δ2 isoforms across leukocyte populations, together with PD-1 and CTLA-4 expression’

One histogram with all cell types presenting FOXP3 expression (in percentage), for both, total FOXP3 expression and both isoforms can be added.

We have included an additional summary figure (Fig. 14) displaying the percentage of FOXP3-positive cells and the distribution of the analyzed FOXP3 isoforms across all leukocyte subsets

 

For Discussion:

it would be more informative if comparison of percentage of FOXP3 + cells among Treg CD4CD25 in other published results from control samples and explanation of their relatively low percentage is added;

Thanks for the comment. It our work FOXP3-FL was detected in around 30 % of CD4+CD25+ T cells population. Some studies indicate that the majority (> 90 %) of CD4+CD25+ cells with high expression of CD25 are FOXP3+ [1]. However, if FOXP3 expression is assessed on all the CD4+CD25+ T cells, independent of level of CD25 expression, between 27 and 52.7% of the cells were FOXP3+ [2], this coinciding with our results. This has been acknowledged in the discussion section of the revised manuscript.

in the discussion it is not accented that CD4CD25 belong to Treg;

It has been included in the discussion

The biological significance of FOXP3 expression in non-CD4 population and hypotheses of the possible consequences of their expression can be discussed;  the role of FOXP3 and its variants in Treg development; role of cell signaling network in the outcomes of FOXP3 expression, functions of deleted form of FOXP3, possible mechanisms of their translation regulation.

We have incorporated a more detailed discussion of the potential biological implications of FOXP3 expression in non-CD4+ cells, including possible regulatory functions, activation-dependent expression, and context-dependent effects on immune responses. Furthermore, we discuss current knowledge regarding the distinct functions of FOXP3 splice variants and the potential influence of signaling pathways and post-transcriptional regulatory mechanisms on FOXP3 expression. We have included the following paragraphs:

Introduction:

- Its expression is regulated by a complex network of signaling pathways involving T-cell receptor activation, IL-2/STAT5 signaling, TGF-β-mediated pathways, epigenetic modifications, and enhancer elements within the FOXP3 locus [9]…

- The exact molecular mechanism of the alternative splicing of Foxp3 pre-mRNA is un-known. However, T cell receptor (TCR) activation can significantly promote Foxp3Δ2 formation [10] FOXP3 splicing isoforms show distinct abilities in the nuclear translo-cation, the DNA-binding ability and the cofactor interaction, resulting in different re-sponses on differentiation, cytokine secretion, suppressive function and linage stabil-ity, of Tregs [11]…

- The loss of FOXP3 exon 2 can also enhance the DNA-binding ability [11].

- It has been proved that the expression of FOXP3-Δ2 is associated with promotion of CD8 T cell–mediated antitumor immunity, this providing resistance to the develop-ment of multiple types of tumors [16]. Increased or imbalanced expression of ΔE2 in relation to FL is often linked to Treg dysfunction and autoimmunity.

- Regulatory T cells exert suppressive functions through multiple mechanisms, including the expression of immune checkpoint molecules such as CTLA-4, PD-1, TIGIT, and LAG-3, many of which are directly or indirectly regulated by FOXP3 [17,18].

- Besides the suppressive role of FOXP3 in canonical CD4+CD25+ Treg cells, experi-mental studies have further demonstrated that ectopic expression of FOXP3 in conven-tional CD4+CD25− T cells can confer suppressive properties and induce the expression of several Treg-associated markers (GITR, CTLA-4, and TGFβ), suppress the prolifera-tion of naïve CD4+T cells and inhibit the activation-induced IL-2 gene expression and the production of IFNϒ in response to TCR ligation [19], this supporting its central role in establishing the regulatory T-cell phenotype

Discussion

- One of the most intriguing observations of the present study is the detection of FOXP3 expression in leukocyte populations beyond the conventional CD4+CD25+ regulatory T-cell compartment.

- Although FOXP3 is widely recognized as the defining transcription factor of Tregs, in-creasing evidence suggests that its expression is not strictly restricted to these cells.

- FOXP3 expression has been reported in activated CD8+ T cells, B lymphocytes, NK cells, and monocyte-derived populations, although its expression levels are generally lower and often more transient than those observed in classical Tregs. The biological significance of this expression remains incompletely understood. One possibility is that FOXP3 participates in activation-induced feedback mechanisms that limit exces-sive immune responses. Alternatively, FOXP3 expression in non-CD4+ populations may reflect context-dependent regulatory programs that become relevant under in-flammatory or pathological conditions.

- The relatively low expression levels observed in some leukocyte subsets raise the possibility that FOXP3 may exert subtle regulatory effects that are highly dependent on cellular context, cofactor availability, and signaling networks. Therefore, the presence of FOXP3 transcripts should not necessarily be interpreted as evidence of a fully de-veloped regulatory phenotype but rather as an indication of potential participation in broader immune regulatory mechanisms. Further functional studies will be required to determine whether the detected FOXP3 isoforms contribute directly to cellular behav-ior in these populations.

 

Can data from RNAseq from different types of leukocytes be extracted and compared with obtained data?

We thank the reviewer for this interesting suggestion. However, RNA-seq data were not generated for the samples included in the present study, and therefore a direct comparison between our results and transcriptomic profiles from different leukocyte subsets cannot be performed. We agree that integrating our findings with publicly available RNA-seq datasets could provide additional mechanistic insights and help further characterize cell type-specific expression patterns. We have acknowledged this point in the Discussion as a limitation of the current study and as a potential direction for future research by stating the following sentence:

- ‘Integrating our findings with RNA datasets could help further characterize cell type-specific expression patterns; however, the scope of the present study did not include transcriptomic analyses, and no RNA-seq data are available from our samples. Future studies integrating transcriptomic approaches may provide additional mechanistic insight’

Other comments

signs + and – can be bigger in the figures.

It has been modified within the new version of the manuscript

In figure legends a number of samples can be given

This information has been added within the first paragraph of results sections in the new manuscript

line  336 vent ratio?

It has been corrected

line 447: sentence reorganization

It has been modified accordingly

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

Hi. Thanks for the effort to improve the paper. While there is a percentage of improvement. Still trying to round out the major results, for that, you only need at most 4 figures. If you are convinced that this study is necessary, in the discussion, start with the main results, FOXP3 in CD4+CD25+ T cells versus PBMCs, for example, the most striking difference. Then, that is the first study characterizing healthy individuals, and then the implication, the clinical significance. It is valid to propose using a scheme. Then the perspective instead of limitations. 

I would help that the title remarks that this is the first study, something that highlights the scientific soundness and usefulness. Avoid justifying in the discussion. 

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

Dear Authors,

 Hi. Thanks for the effort to improve the paper. While there is a percentage of improvement. Still trying to round out the major results, for that, you only need at most 4 figures. If you are convinced that this study is necessary, in the discussion, start with the main results, FOXP3 in CD4+CD25+ T cells versus PBMCs, for example, the most striking difference. Then, that is the first study characterizing healthy individuals, and then the implication, the clinical significance. It is valid to propose using a scheme. Then the perspective instead of limitations. I would help that the title remarks that this is the first study, something that highlights the scientific soundness and usefulness. Avoid justifying in the discussion. 

We greatly appreciate the reviewer's careful and detailed review of our manuscript. The valuable comments and suggestions have helped us improve the quality and presentation of the work. We have tried to address all the concerns raised:

  Title

We have modified the title to highlight the soundness of the manuscript, stating that this is the first study. The current title is: ‘First Comprehensive Analysis of Full-Length and Δ2 Foxp3 Isoforms Distribution in PBMCs from Healthy Volunteers’

 

Discussion

- We have re-phrased the first paragraph to underline our main findings. This is the current version: ‘In this study, we systematically characterized for the first time the distribution of FOXP3 isoforms across human peripheral blood immune cell populations and examined their association with CTLA-4 and PD-1 expression. Although FOXP3 expression has been extensively characterized in regulatory T cells (Tregs), its distribution across the broader hematopoietic compartment remains poorly defined.

The principal finding of this study is that FOXP3-FL predominates in CD4+CD25+ T cells, whereas FOXP3-Δ2 is preferentially detected in other peripheral immune cell populations, including CD8+ T cells, monocytes, and neutrophils, although at low frequencies. In particular, we show that: a) FOXP3 isoform balance varies markedly across immune cell populations; b) the highest FOXP3 expression was observed in CD4⁺CD25⁺ T cells (canonical Treg cells); c) FOXP3-FL was detected in approximately 30% of the CD4+CD25+ T-cell population; d) the full-length isoform (FOXP3-FL) predominated over FOXP3-Δ2, with a FL⁺/Δ2⁺ ratio above three.’…

…‘The simultaneous detection of different isoforms within the same cell may help improve the prediction of the final cellular response. In this context, we analyzed isoform ratios, as a unified predictive parameter that we speculate could be useful both in autoimmune disease scenarios and in therapy-induced immune responses.’

 

- We have also re-phrased the limitations section with a ‘perspectives’ approach. This is the current version: ‘This study establishes a baseline framework for the characterization of FOXP3 isoform distribution across peripheral blood mononuclear cell (PBMC) subsets in healthy individuals and provides a cytometry-based approach for the simultaneous evaluation of FOXP3-FL and FOXP3-Δ2 expression. The relatively small cohort of healthy donors and the low frequency of FOXP3⁺ events, particularly outside the CD4⁺ compartment, highlight the importance of expanding these analyses in larger and more diverse populations to further refine and validate the observed expression pat-terns. A key objective of this work was to map the distribution of FOXP3 isoforms across PBMC populations and to explore potential coordinated patterns of expression that may reflect underlying regulatory imbalances. In this context, future studies assessing FOXP3 isoforms under conditions of immune activation or disease will be instrumental in elucidating their biological and translational relevance, particularly in non-CD4⁺ immune cell subsets where their functional significance remains to be fully defined. Since Foxp3 isoforms exert pleiotropic effect on different factors and can modulate the microenvironment, the FL/Δ2 ratio calculation could be a more valuable predicting parameter; this must be validated in further studies with immune activation, though.

In addition, integrating isoform-level protein expression with transcriptomic da-tasets, including single-cell RNA sequencing approaches, represents a promising ave-nue to further resolve cell type–specific FOXP3 expression programs. Although such analyses were beyond the scope of the present study and no RNA-seq data were available, future multi-omics integration may provide deeper mechanistic insight into FOXP3 regulation across immune compartments.The influence of biological variables such as sex on FOXP3 expression patterns also represents an important area for future investigation. While current evidence remains heterogeneous and sometimes conflict-ing [39, 40], incorporating such parameters in larger cohorts may help clarify their con-tribution to inter-individual variability.

Finally, the associations observed between FOXP3 isoforms and checkpoint mol-ecule expression should be interpreted as descriptive findings that open new hypoth-eses regarding low-level regulatory programs outside the conventional Treg compart-ment. Future functional studies will be essential to determine the biological signifi-cance of these observations and to establish whether they reflect active regulatory mechanisms or context-dependent expression signatures.’

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of the manuscript "Distribution of full-length and Δ2 variants of Foxp3 in PBMCs of healthy volunteers" responded to the comments and improved the content. There are only some minor comments to the manuscript:

regarding small introduction in the Results, although the authors cited it in the response, I did not find it in the manuscript

Tables should have explanations of the abbreviations and bigger + and – in the first column.

lines 23, 488, 565, 599, 84 (thus):  sentence reorganization

The authors added Figure 14 as an overview in the Discussion, but, in general, figures are not common in Discussion, they should be in the Results (and the explanation).

Author Response

Comments and Suggestions for Authors

The authors of the manuscript "Distribution of full-length and Δ2 variants of Foxp3 in PBMCs of healthy volunteers" responded to the comments and improved the content. There are only some minor comments to the manuscript.

We sincerely thank the reviewer for their thorough and detailed evaluation of our manuscript. The comments and constructive suggestions have been invaluable in improving the quality, clarity, and scientific rigor of this work. We have carefully considered all of their observations and incorporated the corresponding revisions, which have significantly strengthened the manuscript.

Regarding small introduction in the Results, although the authors cited it in the response, I did not find it in the manuscript

The reviewer is right, we are sorry for the mistake. It has been included within the new version of the manuscript.

Tables should have explanations of the abbreviations and bigger + and – in the first column.

We have modified them accordingly

lines 23, 488, 565, 599, 84 (thus):  sentence reorganization

Thanks for the suggestion, the sentences proposed have been reorganized.

The authors added Figure 14 as an overview in the Discussion, but, in general, figures are not common in Discussion, they should be in the Results (and the explanation).

The reviewer is right, we thought to include it in the discussion section as a summary of the results, but it has been moved to the end of the results section instead.