CD4 + T cells promote fibrosis during metabolic dysfunction-associated steatohepatitis

  1. Lucía Valenzuela-Pérez
  2. Hyun Se Kim Lee
  3. Rachel L. Bayer
  4. Shravan K. Mishra
  5. Alexander M. Washington
  6. Qianqian Guo
  7. Adam Herman
  8. Rondell P. Graham
  9. Malaz M. Sidahmed
  10. Edward Ssali
  11. Adna A. Hassan
  12. Ece Janet Dinc
  13. Kevin D. Pavelko
  14. Gregory J. Gores
  15. Patrick Starlinger
  16. Xavier S. Revelo
  17. Samar H. Ibrahim
  18. Enis Kostallari
  19. Adebowale O. Bamidele
  20. Petra Hirsova

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Abstract

Unresolved inflammation and fibrosis are the two key features of metabolic dysfunction-associated steatohepatitis (MASH), a progressive form of steatotic liver disease that can evolve into cirrhosis and liver cancer. Although innate immunity has been well studied in MASH, the role of CD4⁺ T cells remains underexplored despite their potential to coordinate immune responses by providing help to other immune cells, promoting inflammation, or regulating immune activity through effector and regulatory subsets. To better understand the role of CD4 + T cells in the pathogenesis of MASH, we comprehensively characterized hepatic CD4 + T cells in murine and human MASH at a single-cell protein, transcriptional, and functional level. Mass cytometry and CITE-sequencing revealed a marked shift in intrahepatic CD4⁺ T-cell composition in MASH, with enrichment of Th1, regulatory, and cytotoxic CD4⁺ T cells. Similar phenotypic changes were mirrored in the peripheral blood and validated in human MASH samples. Functional assays demonstrated increased production of IFNγ and TNFα by hepatic CD4⁺ T cells, highlighting their proinflammatory effector activity. Transcriptomic profiling identified Tnfrsf4 (OX40) upregulation in hepatic CD4⁺ T cells during MASH. Therapeutic blockade of the OX40L-OX40 axis reversed hepatic fibrosis and improved histologic disease scores in mice with established MASH, and also decreased inflammatory markers in a human ex vivo liver model. Together, these studies provide a proteogenomic single-cell atlas for hepatic CD4⁺ T cells and uncover a CD4⁺ T cell-dependent immunopathogenic circuit as a promising immunotherapeutic target to alleviate MASH and liver fibrosis.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a Structured PREreview. You can view the complete PREreview at https://prereview.org/reviews/17220013.

    Does the introduction explain the objective of the research presented in the preprint? Yes The introduction clearly states the focus of the study. It outlines the focal point on hepatic CD4⁺ T cells in MASH and the investigation of the OX40L–OX40 pathway as a potential therapeutic target.
    Are the methods well-suited for this research? Highly appropriate The research employs a robust multi-modal design (CyTOF, CITE-seq, genetic deletion of CD4, antibody blockage and human precision-cut liver slices) that adheres to best practices and offers a sound basis to make valid conclusions.
    Are the conclusions supported by the data? Highly supported The conclusions go well with the experimental data and give a realistic analysis of the results.
    Are the data presentations, including visualizations, well-suited to represent the data? Highly appropriate and clear The data is easy to interpret because the figures, graphs, and visualizations are well designed, clearly labeled, and communicate key findings.
    How clearly do the authors discuss, explain, and interpret their findings and potential next steps for the research? Very clearly The discussion is insightful and well organized in that it gives clear explanations to the findings, their biological importance and how they contribute to the understanding of MASH immunopathogenesis. The logical steps that the authors provide such as the mechanistic research and clinical translation of OX40L-OX40 targeting are also comprehensive, showing a profound and informative understanding of their findings.
    Is the preprint likely to advance academic knowledge? Highly likely The paper offers substantial novel data on the importance of hepatic CD4+ T cells and the OX40L-OX40 axis in MASH, which offers mechanistic knowledge and a potential therapeutic receptor that makes great contributions to knowledge in liver immunopathology.
    Would it benefit from language editing? No
    Would you recommend this preprint to others? Yes, it's of high quality The preprint is scientifically sound and properly executed and it contains new and impactful insights which are useful to immunology, hepatology and translational medicine researchers.
    Is it ready for attention from an editor, publisher or broader audience? Yes, as it is The article is quite robust and close to publication but would require a few modifications, which include better reporting of sample sizes, more mechanistic findings and public deposition of raw data prior to submission to a journal.

    Competing interests

    The author declares that they have no competing interests.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17220152.

    Summary 

    This preprint features an interesting and thoroughly conducted study on the role of CD4 + T cells in the pathogenesis of steatohepatitis as a consequence of metabolic dysfunction (MASH). The authors showed that CD4+ T-cell activation drives liver inflammation and fibrosis, and that OX40L therapeutic targeting reduces disease phenotypes using a potent combination of methods, including CyTOF, CITE-seq, genetic CD4 deletion, OX40L antibody blockage of the OX40L OX40 axis, and human precision-cut liver sections. The multi-modal design and the presence of human tissue give this work good translational relevance and thus makes this a valuable resource to the field.

    Major strengths

    • Technical rigor and breadth: single-cell proteomics, transcriptomics, functional mouse models, and human PCLS integrate to form a powerful, cross-validated dataset.

    •  Clinical relevance: OX40L inhibition reverses fibrosis in pre-existing disease and lowers the levels of inflammatory signals in human liver slices, which puts OX40 as a practical therapeutic target.

    • Resource value; The CITE-seq atlas of hepatic CD4+ T cells is a valuable addition to the mechanistic studies in future.

    Suggestions

    • Open reporting - Have the number of biological replicates of all mouse experiments and human donors clearly indicated and summarized in a table or extended methods section.

    • Antibody description - Include dose, clone, route, supplier and validation information about the OX40L-blocking antibody in order to have reproducibility.

    • Mechanistic insight – Although there is evidence to indicate that a CD4→macrophage→fibrosis pathway is possible, further evidence (such as co-culture or cytokine-neutralization experiments) would support causality.

    • Human data - The data to support the PCLS results indicate the characteristics of the donor, slice viability, and protein/cytotokine results.

    Overall assessment

    This is a quality study that greatly contributes to the current body of knowledge on immune drivers in MASH and presents an effective treatment option. It will be more appropriate to be published in a formal journal with a few minor amendments, and it will be of high interest to researchers in the hepatology and immunology fields.

    Competing interests

    The author declares that they have no competing interests.

  3. Version published to 10.1101/2025.06.27.660220 on bioRxiv