Tumor Immunology Research: The Exhausted State of CD8+ T Cells in Tumors

In recent years, immunotherapy has achieved significant progress in the field of tumor treatment. However, tracking the behavior of lymphocytes following antigen signals remains challenging. This study aims to address this issue by developing Antigen Receptor Signaling Reporter (AGRSR) mice to evaluate the behavior of active T cells within tumors. This paper provides a detailed introduction to the research background, methods, results, and significance.

Research Background

CD8+ T cell exhaustion is an epigenetically propagated (1, 2), time-increasing (3) permanent low-function state that can prevent harmful immune responses (4, 5). Previous studies using sophisticated parallel experiments (6, 7) and Kaede mice (8, 9) have demonstrated the existence of exhausted CD8+ T cells within tumor tissues. These findings are consistent with reports that CD8+ T cells express CD103, a key resident protein, and are primarily confined to the tumor (10, 11). However, recent studies have observed the egress of CD8+ T cells, including antigen-specific T cells, from tumors (8, 9). Thus, it remains unclear how specific factors regulate CD8+ T cell tumor residence and egress behavior.

Research Motivation and Problem

To better understand the behavior of CD8+ T cells within tumors, the research team developed a novel AGRSR mouse model to track antigen-responsive lymphocytes at specific times and locations. Unlike existing T cell receptor (TCR) transgenic lymphocyte (14-16) and tetramer (17, 18) tracking methods, this system can distinguish when and where T cells receive antigen signals. This new method will help verify the role of antigen signals in regulating CD8+ T cell tumor residence and egress behavior.

Research Source

This paper is authored by Takahashi et al. and published in the journal Science Immunology. The authors come from multiple research institutions, including the University of Cambridge, the University of Tokyo, the University of Liverpool, and the Wellcome Trust Sanger Institute.

Research Methods

Development of AGRSR Mice

The research team created AGRSR mice to evaluate clones receiving concurrent TCR signals, including those in cell-sparse tissues. In AGRSR mice, the Nur77 promoter is active only in T lymphocytes upon TCR binding, driving the equimolar expression of red fluorescent protein (Katushka) and Cre-ERT2 recombinase. By crossing with the Rosa-LSL-EYFP strain, AGRSR-LSL-EYFP mice were produced. In these mice, this transgene system acts as a molecular gate, permanently marking T cells and their progeny that receive TCR and tamoxifen signals.

The research team first crossed the AGRSR strain with the OVA-specific OT-I TCR transgenic strain, whose TCR variant peptide ligands had been characterized. Stimulating the naive CD8+ T cells from these animals in vitro showed that the expression level of Katushka was directly related to the stimulatory activity of each ligand, indicating that Katushka expression depended on the strength of the TCR signal.

Research Process

1. Establishment of Mouse Models: The research team crossed AGRSR mice with Rosa-LSL-EYFP mice to generate AGRSR-LSL-EYFP mice for tracking T cells marked by antigen signals.
2. Antigen Stimulation Experiments: The research team verified the dependence of TCR signals through in vitro and in vivo experiments. In the in vivo experiment, tamoxifen-treated AGRSR mice showed TCR signal-dependent EYFP expression in CD8+ T cells from the spleen and tumors.
3. Tumor Model: Using the YUMMER1.7 melanoma model to study the impact of antigen signals on T cell responses, the team found a significant increase in the frequency of EYFP+ CD8+ T cells within the tumor, indicating a response to tumor antigens.
4. Single-Cell RNA Sequencing and TCR Sequencing: Using single-cell RNA sequencing and TCR sequencing, the research team analyzed EYFP+ T cells from tumors and spleen, finding higher exhaustion scores in CD8+ T cells within tumors.

Data Analysis

The research team utilized various experimental techniques and analytical methods, including:

• Flow Cytometry: Staining T cells with surface antibodies and analyzing EYFP and Katushka expression using flow cytometry.
• Single-Cell RNA Sequencing and TCR Sequencing: Using 10x Genomics’ single-cell RNA sequencing and TCR sequencing for gene expression and TCR sequence analysis of sorted T cells.
• Data Analysis: Analyzing data using R and Python packages, including Seurat, Scanpy, and TrajClust algorithms.

Research Results

Antigen Signal-Captured CD8+ T Cells in Tumor Exhaustion

1. AGRSR Mouse Validation: The research team successfully created AGRSR mice and validated their ability to mark T cells receiving antigen signals in vivo.
2. Tumor Model Study: In the YUMMER1.7 melanoma model, tamoxifen-treated AGRSR mice showed a significant increase in the frequency of EYFP+ CD8+ T cells within tumors, with these cells exhibiting high PD-1 expression, indicating an exhausted state.
3. Single-Cell RNA Sequencing Analysis: The research team found that EYFP+ CD8+ T cells within tumors exhibited higher exhaustion scores and lower cytotoxicity scores, while those in the spleen showed higher cytotoxicity scores.
4. Role of Antigen Signals in the Tumor: The study shows that antigen signals play a role in isolating CD8+ T cell responses within the tumor, keeping exhausted T cells in specific tumor regions, thereby protecting the body from systemic damage.

Research Significance

By developing AGRSR mice, this study provides a new tool for tracking the effects of antigen signals on T cell behavior in vivo. The findings reveal how antigen signals regulate CD8+ T cell residence and exhaustion behavior within tumors by isolating their response. This discovery contributes to a deeper understanding of immune response regulation in the tumor microenvironment, providing important clues for developing new immunotherapy strategies.

Research Highlights

1. Development of a New Tool: The creation of AGRSR mice provides a powerful tool for tracking antigen signals in vivo.
2. Mechanism of Antigen Signal Action: The research reveals how antigen signals regulate CD8+ T cell residence and exhaustion behavior by isolating their response.
3. Regulation of the Tumor Microenvironment: The results provide new perspectives for understanding the regulatory mechanisms of the tumor microenvironment’s immune responses.

This study offers important theoretical and practical foundations for tumor immunotherapy research through innovative experimental methods and detailed data analysis. Future research can further explore how to utilize these findings to optimize immunotherapy strategies and enhance treatment efficacy.