Inducing Immune Tolerance through Peripheral Clonal Deletion Targeting PD-1 Expressing Cells

Background Introduction

The establishment of T-cell receptor (TCR) repertoire through thymic negative selection is a key process for achieving self-tolerance and acquired tolerance post-organ transplantation. However, it remains unclear whether the specific mechanisms of peripheral clonal deletion can induce transplant tolerance. This study aims to explore the feasibility and mechanisms of inducing immune tolerance by targeting cells expressing Programmed Cell Death Protein 1 (PD-1). PD-1 is a prominent surface receptor involved in T-cell activation and exhaustion, significantly contributing to chronic infections and tumor immune evasion.

Following organ transplantation, PD-1 is considered a surface marker of reactive T cells and is associated with clonal expansion after encountering alloantigens. This study aims to reshape the TCR repertoire by deleting PD-1 positive cells using custom and antibody methods and to investigate if it can promote immune tolerance in a murine transplantation model.

Origin and Author Information

This paper was co-authored by Jikai Cui, Heng Xu, Jizhang Yu, Shuan Ran, Xi Zhang, Yuan Li, Zhang Chen, Yuqing Niu, Song Wang, Weicong Ye, Wenhao Chen, Jie Wu, Jiahong Xia, among others, from the Department of Cardiovascular Surgery and Transplantation Laboratory of Tongji Hospital affiliated with Tongji Medical College of Huazhong University of Science and Technology, and Houston Methodist Hospital. The research article is published in the April 26, 2024, issue of the journal Science Immunology.

Research Content

Research Process

Overview of Experimental Process

The study initially utilized murine global antigen mismatch (BALB/c to B6) and genetically identical (B6 to B6) heart transplantation models and transferred TCR transgenic CD4+ cells to identify and study specific surface markers of reactive T cells. Through flow cytometry, RNA sequencing, and genetically edited mouse models, the study investigated clonal expansion of reactive T cells and the influence of PD-1 expression on transplant tolerance.

Specific Experimental Steps

1. Screening Markers for Reactive T Cells: On the 6th day post-transplantation, flow cytometry and RNA sequencing were used to analyze TEA TCR transgenic CD4+ cells in the recipient spleen of the BALB/c-to-B6 heart transplant model to assess the expression of these cells’ surface markers.

2. PD-1 Expression in Endogenous Immune Cells: Single-cell RNA sequencing was conducted on T cells from iso-spleen and allo-spleen to identify PD-1 expression levels.

3. Analysis of Clonal Expansion of Endogenous Reactive T Cells: Using single-cell transcriptome and TCR sequencing, the correlation between PD-1 expression and clonal expansion in reactive T cells post-transplant was assessed.

4. Functional Phenotype Analysis of PD-1 Positive Cells: Proliferation and effector molecule expression in PD-1 positive T cells were detected to evaluate their function in reactive T cells.

Data and Results Analysis

RNA Sequencing and Data Analysis: Through principal component analysis (PCA) and gene enrichment analysis, significant upregulation of PD-1 in reactive T cells was identified and verified at the protein level by flow cytometry.

Expression and Functional Analysis of PD-1 in Endogenous T Cells: Single-cell RNA sequencing and TCR sequencing revealed that highly clonally-expanded CD8+ T cells enriched cytotoxic function-related molecules while expressing PD-1.

Main Research Results

1. PD-1 as a Surface Marker of Reactive T Cells: PD-1 expression increased significantly in TEA TCR transgenic CD4+ cells encountering alloantigens, with no significant changes in other marker expressions.

2. Clonal Expansion of Reactive T Cells Correlates with PD-1 Expression: Single-cell RNA sequencing and TCR sequencing indicated that highly clonally expanded T cell populations showed stronger cytotoxic functions while expressing PD-1.

3. Deletion of PD-1 Positive Cells Promotes Transplant Tolerance: Using the PD-1dtr mouse model, DTR-mediated cell elimination significantly prolonged the survival time of skin and heart transplants, and delayed graft rejection was observed post deletion of graft-infiltrating PD-1 positive T cells.

4. Analysis of Residual T Cell Response Specificity: In vitro experiments showed significantly reduced responses of residual T cells in recipients to BALB/c antigens, indicating that elimination of PD-1 positive cells mainly targets specific allo-reactive T cells rather than global immune suppression.

Conclusion and Significance

The study demonstrates the critical role of PD-1 in reactive T cells and suggests that targeting and deleting PD-1 positive cells can reshape the TCR repertoire through peripheral clonal deletion, thereby promoting immune tolerance in murine transplantation models. This method holds promise for adjusting immune responses in transplant recipients and may serve as a potential strategy for treating autoimmune diseases.

Research Highlights

• Innovative Technology: This study employed DTR mechanism and specific antibody deletion strategies to verify the importance of PD-1 positive cells in transplant immune tolerance.
• Scientific Value: By revealing PD-1 as a marker for reactive T cells and reshaping the TCR repertoire in transplant and autoimmune settings, this study provides new theoretical basis for targeted immune regulation.
• Clinical Significance: The research indicates that this immune regulation strategy may be applicable in clinical transplantation and for treating autoimmune diseases, helping reduce infection risks associated with traditional immunosuppressive therapies.

Through comprehensive experimental data and analysis, this paper detailed the role and potential applications of PD-1 targeting deletion in inducing immune tolerance, providing a rich scientific foundation for future research and clinical applications.