Interaction and Attenuation of Tumor-Specific CD8+ T Cell Responses by LRIG1 and VISTA

Academic Background

In recent years, the importance of immune checkpoint inhibitors (ICIs) such as Programmed Cell Death Protein 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) has received increasing attention. These inhibitors significantly improve the survival of some cancer patients by enhancing anti-tumor T cell responses. However, the overall response rate of existing ICIs therapies remains low, necessitating the identification of new immune checkpoints as alternative therapeutic targets.

Studies have identified a class of “stem cell-like” tumor-specific CD8+ T cells expressing T-cell factor 1 (Tcf-1), which respond significantly to ICIs therapy. These “stem cell-like” T cells undergo proliferative bursts upon exiting a quiescent state. However, the mechanisms maintaining Tcf-1+ stem cell-like T cells in a quiescent state remain unclear, which could potentially cause resistance to existing ICIs therapies observed clinically.

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an immune checkpoint protein in the B7 family and is considered a potential target for next-generation immunotherapy. Despite VISTA being thought to suppress T cell activation in two ways, its downstream signaling mechanisms remain unclear.

This study aims to identify the inhibitory receptor LRIG1 (Leucine-rich repeats and Immunoglobulin-like domains 1) that interacts with VISTA and to investigate how this interaction controls anti-tumor immune responses.

Source of the Paper

This paper was authored by Hieu Minh Ta and colleagues from the Cleveland Clinic Taussig Cancer Institute, Lerner Research Institute, and Case Western Reserve University. It was published in the journal Science Immunology on May 17, 2024.

Research Workflow

Identifying LRIG1 as a VISTA Binding Receptor

This study first identified LRIG1 as a VISTA binding receptor using Selective Protein Proximity Labeling and Tagging (SPPLAT) under neutral pH conditions. Mass spectrometry identified LRIG1 as a VISTA-binding ligand, and several experiments were conducted to verify the interaction between VISTA and LRIG1, including surface plasmon resonance (SPR) assays and co-immunoprecipitation (Co-IP) tests.

Experimental Steps:

1. SPPLAT Assay: Conducted proteomic analysis using activated wild-type mouse T cells and VISTA knockout mouse spleen T cells to identify and verify the interaction between VISTA and LRIG1.
2. Surface Plasmon Resonance (SPR) Assay: Further confirmed the binding affinity of VISTA and the extracellular domain (ECD) of LRIG1 under neutral and acidic conditions.
3. Luciferase Binding Assay: Detected the interaction between VISTA and LRIG1 on the T cell surface, including analyzing “cis” interactions (binding within the same cell) and “trans” interactions (binding between different cells) between VISTA and LRIG1.

Inhibitory Effects of LRIG1 on T Cell Signaling

To investigate the function of LRIG1 in T cells, the study elucidated the impact of LRIG1 on T cell receptor (TCR) signaling pathways through the following experimental steps and verified the phenotypes of LRIG1-deficient mice. 1. Mouse Model Experiments: Generated mice carrying LRIG1 knockout alleles and crossbred them with mice expressing the OT1 TCR transgene. Assessed the impact of LRIG1 deficiency on TCR signaling by stimulating naive OT1 CD8+ T cells. 2. Phosphorylation Analysis: Examined the phosphorylation levels of intracellular signaling molecules (LAT, PLC-γ, SLP76, AKT, ERK1/2), demonstrating the necessity of the LRIG1 cytoplasmic domain in relaying inhibitory signals.

Effects of LRIG1 on T Cell Proliferation, Survival, and Effector Function

By stimulating LRIG1-deficient OT1 T cells, their proliferation, cytokine secretion, and apoptosis status were detected. Results showed that LRIG1-deficient T cells had enhanced expansion and effector functions. Further, resistance to VISTA-mediated inhibition was explored through coculture experiments, and these results were validated in in vivo tumor models.

Single-Cell Transcriptome Analysis

To further analyze the heterogeneity of tumor-infiltrating CD8+ T cells (TILs), the study conducted single-cell RNA sequencing (scRNA-seq) analysis. Multiple subsets related to T cell activation and functional states were identified, and the performance of LRIG1-deficient and wild-type T cells in the tumor microenvironment was compared.

Validation with Human Cancer Samples

Analysis of TILs in human melanoma, endometrial cancer, and lung cancer samples revealed that LRIG1 was expressed in various activated states of T cells. Higher levels of LRIG1 expression correlated with resistance to immunotherapy.

Research Results

The results indicated that LRIG1, in conjunction with VISTA, inhibits anti-tumor responses both intracellularly and extracellularly in T cells. LRIG1-deficient mouse T cells exhibited enhanced signaling, proliferation, and effector functions and showed stronger anti-tumor responses in tumor models. Additionally, high levels of LRIG1 in human cancer samples were associated with resistance to immune therapy, highlighting the potential application of LRIG1 as an immune checkpoint receptor in cancer treatment.

Conclusions and Significance

The interaction between LRIG1 and VISTA provides new insights into the inhibitory mechanisms of VISTA in T cells. As a binding receptor for VISTA, LRIG1 not only inhibits T cell responses in vivo and in vitro but also affects the efficacy of tumor immunotherapy. This study demonstrates the potential application of targeting the VISTA/LRIG1 axis as a cancer immunotherapy strategy, providing essential theoretical foundations for developing new immunotherapies.

Research Highlights:

1. Identification of a New Target: Confirmed LRIG1 as a VISTA binding receptor for the first time and revealed its role in T cell inhibition.
2. Mechanism Elucidation: Detailed the mechanisms by which VISTA and LRIG1 inhibit T cell signaling through “cis” and “trans” interactions.
3. Clinical Relevance: Discovered that high levels of LRIG1 expression are associated with resistance to ICIs, offering new directions for developing cancer treatments.

This research lays a solid foundation for future development of new cancer immunotherapies, underscoring the potential importance of LRIG1 as an immune checkpoint receptor.