Induction of Tertiary Lymphoid Structures in Glioma Immunotherapy and Their Anti-Tumor Immune Effects

Background Introduction

Glioma is a highly malignant brain tumor characterized by limited lymphocyte infiltration in the tumor microenvironment (glioma microenvironment, GME), presenting an “immune desert” state. This characteristic makes glioma insensitive to various immunotherapies, leading to poor treatment outcomes. In recent years, immunotherapy has made significant progress in the treatment of various solid tumors, but its application in glioma still faces significant challenges. Although immunotherapy research targeting glioblastoma (GBM) has been conducted, less than 20% of patients achieve durable clinical benefits. Therefore, exploring new immunotherapy strategies to promote tumor-infiltrating lymphocyte (TIL) infiltration in GME and transforming GME from an immune-resistant to an immune-activated state has become a focus of current research.

Tertiary lymphoid structures (TLS) are structures formed in the tumor microenvironment that resemble secondary lymphoid organs and can promote local immune responses. The presence of TLS is associated with improved prognosis in various cancers, and inducing TLS formation in immune “cold” tumors is considered a promising therapeutic strategy. However, the role of TLS in the glioma microenvironment and its induction mechanisms remain unclear. Therefore, this study aims to explore strategies for inducing TLS formation through Toll-like receptor (TLR) agonists to enhance anti-glioma immune responses.

Source of the Paper

This paper was co-authored by Shaoping Shen, Yong Cui, Mingxiao Li, and others, with the primary authors affiliated with Beijing Tiantan Hospital Neurosurgery Department and Capital Medical University, among other institutions. The paper was published online in Neuro-Oncology on August 27, 2024, under the title “Toll-like receptor agonists promote the formation of tertiary lymphoid structure and improve anti-glioma immunity.”

Research Process and Results

1. Study Design and Experimental Process

This study induced TLS formation in GME through intracranial injection of TLR agonists (OK-432) and glioma antigens (i.c. αTLR-mix) and explored its mechanisms in anti-glioma immunity. The research was divided into the following main steps:

a) Establishment and Treatment of Glioma Mouse Models

• Study Subjects: C57BL/6 mice were used, and GL261 and CT-2A glioma cells were implanted to establish intracranial glioma models.
• Treatment Methods: TLR agonists and glioma antigens were mixed and administered via intracranial injection (i.c.) to the treatment group, while the control group received PBS.
• Experimental Design: Mice in the treatment and control groups underwent MRI scans on days 7, 14, and 21 post-treatment to assess tumor growth. Brain tissue samples were collected on day 21 for further analysis.

b) Detection and Analysis of TLS Formation

• Staining Analysis: Hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), and multiplex immunofluorescence (mIHC) were used to detect TLS formation and its cellular composition.
• Flow Cytometry: Infiltration of B cells (B220+), T cells (CD3+ CD4+, and CD3+ CD8+) in brain tissues was analyzed.
• Single-Cell RNA Sequencing (scRNA-seq): Single-cell RNA sequencing was performed on brain tissue and cervical lymph node (CLN) samples to analyze the state and differentiation trajectories of B and T cells.

c) Exploration of TLS Induction Mechanisms

• Antibody Neutralization Experiments: The role of TNF-α and LTβR signaling pathways in TLS formation was evaluated by injecting blocking antibodies.
• Cell-Cell Interaction Analysis: CellPhoneDB software was used to analyze scRNA-seq data and identify key cell-cell interactions during TLS induction.

d) Preclinical Studies

• Clinical Trial: A clinical trial titled “Combined Stereotactic Radiosurgery and Enhanced Immunotherapy for rGBM” was conducted to evaluate the safety and preliminary efficacy of i.c. αTLR-mix treatment.

2. Main Research Findings

a) TLS Formation and Lymphocyte Infiltration

• TLS Formation: TLS formation was successfully induced in the GME of treatment group mice, consisting mainly of CD19+ B cells, CD3+ CD4+/CD8+ T cells, and CD11b+ myeloid cells.
• Increased Lymphocyte Infiltration: Infiltration of B and T cells in the GME of treatment group mice significantly increased, particularly CD4+ T cells.

b) Mechanisms of TLS Induction

• Role of LTα/β-LTβR Signaling Pathway: Antibody neutralization experiments revealed that the LTα/β-LTβR signaling pathway plays a key role in TLS induction. Blocking this pathway significantly inhibited TLS formation and accelerated tumor growth.
• Role of TH17 Cells: scRNA-seq data indicated that CD4+ TH17 cells functioned as “lymphoid tissue inducer” (LTI) cells in TLS induction.

c) Role of TLS in Anti-Glioma Immunity

• Key Role of CD4+ T Cells: Antibody neutralization experiments showed that CD4+ T cells play a crucial role in TLS formation and anti-glioma immunity, while CD8+ T cells also contribute to anti-tumor immunity, but B cells have a less significant role.
• Differentiation and Clonal Selection of B Cells: scRNA-seq and BCR sequencing analysis revealed that B cells in brain tissues underwent a transition from naïve to mature states, exhibiting clonal selection and class switch recombination (CSR) characteristics.

d) Preclinical Study Results

• Preliminary Clinical Trial Results: In the clinical trial, i.c. αTLR-mix treatment showed good tolerability, with tumor volume reduction in some patients and significantly prolonged progression-free survival (PFS) and overall survival (OS) in the treatment group.

3. Research Conclusions and Significance

This study demonstrates that inducing TLS formation in GME through TLR agonists can significantly enhance anti-glioma immune responses, improve the immune microenvironment, and control tumor growth. TLS may serve as a site for antigen presentation in GME, promoting the activation of local immune responses. This finding provides new insights and experimental foundations for glioma immunotherapy.

4. Research Highlights

• New Strategy for TLS Induction: This study is the first to successfully induce TLS formation in GME using TLR agonists and elucidate its anti-tumor immune mechanisms.
• Key Role of TH17 Cells: The study found that TH17 cells function as “lymphoid tissue inducer” cells in TLS induction, providing a new perspective for understanding TLS formation mechanisms.
• Potential for Clinical Translation: Preliminary results from the clinical trial suggest that i.c. αTLR-mix treatment has potential application value in glioma patients.

Summary

Through systematic experimental design and in-depth data analysis, this study reveals the important role of TLS in glioma immunotherapy and provides strong experimental evidence for future clinical translation. This research not only opens new directions for glioma immunotherapy but also offers insights for the treatment of other immune “cold” tumors.