ITK deficiency protects against autoimmune pulmonary hemorrhage by promoting Amphiregulin-producing regulatory T cells

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

In this study, Hossain et al. investigated the role of Interleukin-2-inducible T cell kinase (ITK) in autoimmune lung injury, demonstrating that ITK-deficient (Itk-/-) mice are protected against pristane-induced pulmonary hemorrhage (PH). Authors suggest that this protection correlates with a significant remodeling of the T cell compartment in Itk-/- mice, including increased frequency of memory-like CD4+ and CD8+ T cells (CD44⁺CD62L⁺) as well as higher frequency of Treg populations. Furthermore, adoptive transfer of ITK-deficient Treg isolated from injured ITK-deficient mice confers protection against pulmonary hemorrhage in WT recipients.

Strengths:

The adoptive transfer of congenically marked wildtype and Itk-/- Treg populations clearly demonstrates that ITK-deficient Treg can actively rescue pre-existing lung injury and reverse systemic secondary metrics like proteinuria in wild-type recipients, providing proof-of-concept validation for the therapeutic utility of the ITK-Treg axis.

Reviewer #2 (Public review):

Summary:

In this manuscript, Hossaim and colleagues investigate the role of the ITK kinase in modulating inflammation in a pristane-induced lung hemorrhage model. Using a germline ITK KO mouse, they report that loss of ITK skews the T cell compartment toward a memory-like state, expanding Tregs, and conferring protection against alveolar hemorrhage, inflammatory monocyte recruitment, proteinuria, and systemic cytokine elevation. They further show that transfer of ITK-deficient Tregs into wild-type hosts with established disease attenuates injury and shifts the cytokine balance toward resolution, and that ITK-deficient Tregs carry a transcriptional signature enriched for OXPHOS, mTORC1, MYC, and cell-cycle programs. While these observations are interesting for the development of potential immunotherapies, there are several issues with the methodological approach that support the authors' claims, tempering my enthusiasm for this manuscript.

Strengths:

(1) The clinical motivation and potential targeted therapies are relevant.

(2) The murine phenotype seems robust.

Comments on revised version.

The authors have addressed my comments appropriately.

Reviewer #3 (Public review):

Summary:

Hossain et al. investigate the role of ITK as a central regulator of autoimmune lung injury. They used ITK-deficient mice and the pristane-induced pulmonary hemorrhage (PH) model to show that ITK deficiency confers protection against PH. The adoptive cell transfer experiment suggests a possible role for altered Treg cells in ITK-deficient mice in regulating the inflammatory response in the lungs of pristane-injected mice. This study shows that targeting the ITK axis may be beneficial by reducing systemic inflammatory injury that contributes to poor outcomes in PH.

Strengths:

This study highlights the importance of ITK in regulating pulmonary hemorrhage. The enrichment of Treg cells is known to confer protection in autoimmunity-mediated alveolar damage. However, the involvement of ITK in regulating the Treg cells' function is interesting and could be explored as a novel therapeutic approach for chronic inflammation.

Comments on revised version:

The authors have answered my comments by incorporating new results and clarification to their observation.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Strengths:

The adoptive transfer of congenically marked wildtype and Itk-/- Treg populations clearly demonstrates that ITK-deficient Treg can actively rescue pre-existing lung injury and reverse systemic secondary metrics like proteinuria in wild-type recipients, providing proof-of-concept validation for the therapeutic utility of the ITK-Treg axis.

We sincerely thank Reviewer #1 for recognizing the strength and translational significance of our adoptive-transfer experiments. We agree that these studies provide important proof-of-concept evidence that ITK-deficient Tregs are not simply associated with protection but can actively ameliorate established pulmonary hemorrhage and associated systemic manifestations when transferred into wild-type recipients. We appreciate the reviewer’s recognition of the potential therapeutic significance of targeting the ITK–Treg axis.

Weaknesses:

The molecular analysis of Tregs isolated from unchallenged wild-type ITKdeficient mice has limited value to understand how the ITK-deficient Tregs protect against autoimmune pulmonary hemorrhages in lungs. Detailed molecular analysis of Tregs isolated from the lung will be helpful in understanding the mechanistic regulation and identifying further therapeutic targets.

We thank the reviewer for this important observation and agree that detailed molecular characterization of lung-resident Tregs following pristane-induced pulmonary injury would provide additional insight into the mechanisms responsible for protection.

The transcriptional analyses presented in the current manuscript were designed to identify intrinsic molecular differences associated with ITK deficiency in Tregs independent of the inflammatory environment. These studies revealed alterations in pathways including oxidative phosphorylation, mTORC1 signaling, MYC-associated programs, and cell-cycle regulation, thereby providing a molecular framework for the enhanced function of ITK-deficient Tregs.

We recognize, however, that the phenotype and transcriptional program of Tregs within the injured lung microenvironment may differ from those observed under baseline conditions. We therefore consider direct molecular comparison of lung Tregs isolated from pristane-treated wild-type and ITK-deficient mice an important direction for subsequent mechanistic studies.

Importantly, the principal functional conclusion of the current study is supported by the adoptive transfer experiments shown in Figure 5. Transfer of ITK-deficient Tregs into wild-type mice with established disease significantly reduced pulmonary hemorrhage and systemic injury, demonstrating that ITK-deficient Tregs themselves are capable of mediating protection. We have clarified this limitation and the need for future studies examining lung-resident Tregs in the revised Discussion. See page 21 line 353- 361.

Reviewer #2 (Public review):

The authors have addressed my comments appropriately.

We sincerely thank Reviewer #2 for carefully evaluating our revised manuscript and for concluding that our previous responses appropriately addressed the reviewer’s concerns. We also appreciate the reviewer’s recognition of the potential relevance of our findings for the development of targeted therapeutic approaches.

Reviewer #2 (Recommendations for the authors):

I would strongly recommend removing Fig. 5J (IF), as there is no convincing signal for Foxp3. This is a well-known issue with Foxp3 Ab. If authors would like to include imaging, would recommend performing smFISH/RNAScope.

We thank the reviewer for this helpful recommendation. We agree that the Foxp3 immunofluorescence signal in the previous Figure 5J does not provide sufficiently convincing visualization to support our conclusions. We have therefore removed Figure 5J from the revised manuscript.

Importantly, our conclusions regarding the protective activity of ITK-deficient Tregs are independently supported by flow-cytometric analyses and, most importantly, by the adoptive-transfer experiments demonstrating that ITK-deficient Tregs can ameliorate established pulmonary hemorrhage in wild-type recipients.

We appreciate the reviewer’s suggestion regarding smFISH/RNAscope and agree that this approach

could provide valuable spatial information in future studies.

Reviewer #3 (Public review):

The novelty of this study lies in the association between ITK-deficient Tregs and pulmonary hemorrhage in autoimmunity. One limitation of this study is that the authors performed all experiments in ITK global knockout mice, in which not only T cells but all other cell types are deficient in ITK. Therefore, further studies are required for a mechanistic rationale for targeting ITK in severe inflammatory lung disease.

We sincerely thank Reviewer #3 for recognizing the novelty of the relationship between ITK-deficient Tregs and protection from autoimmune pulmonary hemorrhage, as well as the potential therapeutic relevance of this pathway in chronic inflammatory disease.

We agree that the use of a germline ITK-deficient mouse represents a limitation of the current study because it does not allow us to completely distinguish T-cell-intrinsic effects of ITK deficiency from potential contributions of other immune populations.

However, our adoptive-transfer experiments provide important functional evidence supporting a direct role for ITK-deficient Tregs. Specifically, purified ITK-deficient Tregs transferred into wild-type recipients with established pulmonary hemorrhage significantly reduced pulmonary injury and associated systemic manifestations. These findings demonstrate that ITK-deficient Tregs are themselves sufficient to confer substantial protection within an otherwise ITK-sufficient host.

Nevertheless, we agree that future studies using lineage-specific conditional ITK-deficient models will be important to define the Treg-intrinsic contribution of ITK signaling more precisely. Such studies, together with evaluation of ITK modulation in human Tregs and appropriate humanized models, will be important for determining the translational potential of targeting the ITK pathway in severe inflammatory lung disease.

We have revised the Discussion to more clearly acknowledge this limitation and highlight these future directions. We again thank the editors and reviewers for their thoughtful and constructive comments. Their suggestions have substantially strengthened the manuscript and helped us more clearly define both the significance of our findings and the remaining questions that will guide future investigation. See the limitations of this manuscript.