Research Report: Differential Characteristics and Functions of Regulatory T Cells in the Brain and Spinal Cord

Research Background and Motivation

This study explored the characteristics and functional differences of regulatory T cells (Tregs) within the central nervous system (CNS). Tregs play a key role in the adaptive immune response, primarily by suppressing excessive immune reactions by recognizing a variety of self and foreign antigens. Tregs are found in various non-lymphoid tissues, such as fat, skin, lungs, intestine, heart, and brain, known as “Tissue Tregs.” They play an important role in tissue homeostasis and repair through interactions with tissue cells. While there has been significant progress in the study of Tregs, the key factors determining the tissue specificity of Tregs (such as antigen specificity, tissue environment, and pathological conditions) are not clear. This study attempts to elucidate the mechanisms of Tregs specificity characteristics in the CNS by using mouse models of ischemic stroke and experimental autoimmune encephalomyelitis (EAE).

Research Origin

The paper was authored by Mahiro Watanabe, Ako Matsui, Natsumi Awata, Ayame Nagafuchi, Mio Kawazoe, Yoshihiro Harada, and Minako Ito, all from the Department of Allergy and Immunology at Kyushu University’s Institute of Medical Science. The research was published in the “Journal of Neuroinflammation” in 2024, with the citation number 21:146.

Research Methods

Experimental Mice

The study used mouse models, including wild-type C57BL/6J mice and specific genetically modified mice (such as Foxp3^hcd2ki mice, IL-17A-GFP mice, and DEREG mice), all raised in Kyushu University’s specific pathogen-free (SPF) animal facility.

Ischemic Stroke Model

The ischemic stroke experiment used male mice aged 8-12 weeks, weighing 20-30 grams, constructed using a temporary filament for middle cerebral artery occlusion (MCAO) for 60 minutes, followed by removal of the filament for cerebral reperfusion. The neurological function of mice was assessed by a neurological scoring method.

EAE Model

Subcutaneous injection of myelin oligodendrocyte glycoprotein peptide (MOG33-55) mixed with complete Freund’s adjuvant in 2-3-month-old FoxP3HCD2-KI and wild-type mice, followed by intraperitoneal injection of pertussis toxin on days 0 and 2.

CNS Cell Separation

The mice were perfused with PBS, and the brains and spinal cords were removed for mechanical and enzymatic dissociation. Flow cytometry analysis and sorting were performed with fluorescently labeled antibodies to detect a variety of specific proteins and molecular markers.

Immunohistochemistry

Mouse spinal cord sections were fixed with 4% paraformaldehyde (PFA) and immunostained with specific antibodies, such as anti-myelin basic protein (MBP), anti-glial fibrillary acidic protein (GFAP), and anti-ionized calcium-binding adaptor molecule 1 (Iba1).

Tregs Depletion Experiment

In EAE mice, depletion of Tregs was induced using diphtheria toxin (DT), and analysis of the brain and spinal cord was carried out after 7 days.

Tregs Transfer Experiment

Tregs isolated from the brains and spinal cords of EAE mice were transferred to wild-type EAE mice to assess their effects on EAE symptoms.

Chemokine Array and Migration Analysis

Proteins from each tissue were extracted for chemokine array analysis; isolated Tregs were subjected to in vitro migration analysis upon exposure to specific chemokines.

RNA and Single-Cell RNA Sequencing

Massive RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) were conducted using RNA extracted from brain and spinal cord samples, with analysis using specific software such as Cell Ranger and Loupe Browser.

Research Results

The Role of Regulatory T Cells in EAE Mice

The lack of regulatory T cells led to exacerbated symptoms in EAE mice, highlighting the importance of Tregs in suppressing the progression of EAE.

CNS Tregs Gene Expression Analysis

Gene expression analysis of Tregs from the brains and spinal cords of ischemic stroke and EAE mice revealed the tissue characteristics of these cells under different pathological states. PCA analysis showed that brain Tregs in EAE were more similar to brain Tregs in the stroke model than to spinal cord Tregs in EAE. Further analysis showed significant differences in tissue-specific gene expression, indicating that environmental factors are more decisive than the pathological state.

T Cell Receptor (TCR) Analysis and Antigen Specificity

TCR sequencing indicated the presence of highly clonally expanded TCRs in the brain and spinal cord, revealing that the tissue-specific features of Tregs are not entirely dependent on antigen specificity.

Chemokine and Chemokine Receptor Interactions

The expression of chemokine receptors on Tregs varied between tissues, with spinal cord Tregs highly expressing CCR1, CCR2, CXCR6, and CCR8, while brain Tregs highly expressed CXCR4. Differences in the expression of corresponding chemokines in each tissue affected the local positioning and function of Tregs.

The Anti-Inflammatory and Repair Effects of Tregs

Transfer experiments showed that Tregs isolated from the spinal cords of EAE mice significantly alleviated EAE symptoms in the same tissue, whereas the effects of brain Tregs were intermediate. These results indicate that Tregs have tissue-specific effects in suppressing inflammation and promoting tissue repair.

Conclusion and Research Value

This study reveals the differential characteristics and functions of brain and spinal cord Tregs in different pathological environments within the CNS, emphasizing the importance of the tissue environment in determining the characteristics of Tregs. These findings are significant for the development of tissue-specific anti-inflammatory therapies via regulatory T cells. The study also proposes that, despite clear differences in tissue characteristics, the unique reparative factors and anti-inflammatory actions of Tregs have potential therapeutic application prospects.

Research Highlights

1. Demonstrates the differences in properties and functions of brain and spinal cord Tregs within the CNS.
2. Highlights the role of the tissue environment in shaping the specificity of Tregs characteristics, more so than pathological status or antigen specificity.
3. Describes the cell migration pathways and reciprocal interactions of Tregs, providing new perspectives on the potential therapeutic applications of Tregs.

This research delves into the unique characteristics and functions of Tregs in the central nervous system, providing rich data support and research directions for future studies on Tregs-based disease treatment.