LRIG1 engages ligand VISTA and impairs tumor-specific CD8+ T cell responses

Rheumatology and Immunology Medicine Cell Biology Oncology Life Sciences Immunology
LRIG1 VISTA tumor-specific CD8+ T cells immune checkpoint anti-tumor immunity immunotherapy

Immunotherapy Checkpoint Inhibitors (ICIs, also known as immune checkpoint blockers) are a class of drugs that have achieved significant breakthroughs in cancer immunotherapy in recent years. These drugs mainly work by blocking Programmed Death Protein 1 (PD-1) and Cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4), thereby activating anti-tumor T-cell responses. However, the overall response rate to current ICI therapies remains low, and there is an urgent need to find new immune checkpoints as therapeutic targets. Recent studies have established the important role of “stem cell-like” tumor-specific CD8+ T cells in responding to ICI therapy, with these cells functioning by exiting a quiescent state and undergoing proliferative bursts. However, the mechanisms that maintain these “stem cell-like” T cells in a quiescent state are unclear and may be a potential cause of clinical resistance to current immunotherapy.

V-domain Ig Suppressor of T Cell Activation (commonly referred to as VISTA) is an immune checkpoint protein of the B7 family, considered a potential target for the next generation of immunotherapy. Previous studies have shown that VISTA acts as an “inhibitory ligand” by binding to the corresponding receptors on T cells and inhibiting T cell activation. However, the downstream signaling mechanisms of VISTA are not clear, hindering the development of precise VISTA inhibitors and the identification of related biomarkers.

Research Source

The article was written by a collaborative team including Hieu Minh Ta, Dia Roy, Keman Zhang, and Tyler Alban from the Cleveland Clinic, United States, and published in the journal Science Immunology on May 17, 2024. The research was mainly conducted at the Cleveland Clinic Lerner Research Institute’s Center for Immunotherapy and Precision Immuno-Oncology.

Research Process

Research Procedure

The research team carried out a series of experiments to identify VISTA’s binding receptors under neutral pH conditions and to explore its specific role and mechanism among inhibitory receptors. The steps included:

  1. Selective Proteomic Proximity Labeling Assay: A comparative experiment between VISTA knockout and wild-type mouse spleen T cells was performed using Tyramide technology for labeling and tracking protein mass spectrometry analysis.

  2. Mass Spectrometry Analysis: Identified LRIG1, a type I transmembrane protein with 1092 amino acids, as a binding partner of VISTA using mass spectrometry.

  3. Co-immunoprecipitation and Surface Plasmon Resonance Experiments: Expressed VISTA and LRIG1 in HEK 293 cells and validated their interaction using antibody-based Co-Immunoprecipitation (Co-IP) and Surface Plasmon Resonance (SPR) techniques.

  4. Mouse Model Experiments: Conducted anti-tumor response tests using mice with T-cell-specific LRIG1 deficiency, comparing tumor control and the performance of T-cell-specific Cytotoxic T Lymphocytes (CTLs) between these mice and normal mice.

  5. Human Sample Experiments: Measured LRIG1 expression from tumor-infiltrating T cells in melanoma patients to explore its reactivity in the application of immunotherapy.

Experimental Techniques and Innovations

The study employed various innovative techniques and methods, including:

  • Selective Proteomic Proximity Labeling Assay using Tyramide (SPPLAT) for identifying protein interactions.
  • Antibody-based Co-Immunoprecipitation (Co-IP) assay for detecting direct protein interactions within cells.
  • Surface Plasmon Resonance (SPR) for analyzing protein binding characteristics and affinity.
  • Luciferase-based Binding Assay for assessing the interaction of cell surface proteins.

Research Results

Binding and Inhibitory Actions of VISTA with LRIG1

  1. In Vitro Experiments:

    • Used SPPLAT technology to verify the binding of VISTA with the extracellular domain of LRIG1 at pH 7.4 and pH 6.0.
    • Validated the direct binding of VISTA with LRIG1 in HEK 293 cells and mouse spleen T cells through Co-IP experiments.

  2. Mouse Model Experiments:

    • T-cell-specific LRIG1 deficiency in mice led to enhanced tumor-specific CTL functions and increased survival, while simultaneously reducing the number of quiescent CTLs.
    • The VISTA-LRIG1 axis suppressed several T cell receptor signaling pathways, including Linker for Activation of T cells (LAT), Phospholipase C (PLC-γ), SLP76, AKT, and Extracellular Signal-Regulated Kinase (ERK1/2).

  3. Clinical Relevance in Humans:

    • High expression of LRIG1 in tumor-infiltrating CTLs of melanoma patients was found to be associated with resistance to immunotherapy.

Main Findings

  • Identified LRIG1 as the mechanism through which VISTA regulates tumor-specific T cell responses by inhibiting the T cell receptor signaling pathway.
  • Defined the anti-tumor effects of LRIG1 deficiency, enhancing the expansion, survival, and effector function of CTLs.
  • Explored the potential role and significance of LRIG1 in clinical immunotherapy.

Research Conclusion and Value

Through various experimental methods, this paper reveals the key role of LRIG1 in the VISTA signaling pathway. The binding of LRIG1 to VISTA is one of the mechanisms for inhibiting T cell activation. The absence of LRIG1 can significantly enhance anti-tumor immune responses, improving the response rate to immunotherapy.

Scientific and Application Values

  1. Scientific Value: Gained a deeper understanding of the VISTA signaling pathway and its inhibitory mechanism, providing a theoretical basis for the future development of more precise VISTA inhibitors.
  2. Application Value: The study shows LRIG1 as a new therapeutic target, suggesting that targeting this pathway can enhance the effect of clinical anti-tumor immunotherapy.

Research Highlights

  • Newly discovered LRIG1 as a VISTA binding partner: Innovatively identified the binding of LRIG1 to VISTA at neutral and acidic pH, providing a new perspective for further understanding VISTA’s inhibitory mechanism.
  • Significant anti-tumor immune responses: LRIG1-deficient mice exhibited stronger tumor-specific CTL activity, indicating LRIG1 is an important negative regulator.
  • Clinical Relevance: In human tumor samples, the expression of LRIG1 and VISTA was associated with resistance to immunotherapy, suggesting its potential as a biomarker.

Conclusion

This research systematically reveals the key role of LRIG1 in the VISTA signaling mechanism and verifies its function as an immune checkpoint receptor. By exploring the inhibitory pathways of LRIG1, this study provides new therapeutic targets and strategies with significant scientific and clinical implications.

This report summarizes the research on the interaction between VISTA and LRIG1 and their inhibitory mechanisms in anti-tumor immunity published in Science Immunology, providing an important reference for future research in related fields and having a broad application prospect.