Long-term Effects of CD8+ T Cell Subsets in Neuroinflammation After Ischemic Stroke

Background

Ischemic stroke is a leading cause of death and disability. After stroke, neuroinflammation is rapidly induced, activating various cells such as mast cells, astrocytes, microglia, and vascular endothelial cells. These reactive cells then express various inflammatory mediators, guiding leukocytes into the core and surrounding areas of brain lesions (Jayaraj et al., 2019). Among the infiltrating leukocytes, T cells play a significant role in post-stroke neuronal damage under both antigen-dependent and independent conditions (Selvaraj & Stowe, 2017; Zhang et al., 2021). Previous studies have shown that T cell migration and presence in the injured side of the brain significantly affect post-stroke neuroinflammation in both subacute (48 hours to 7 days) and late (>7 days) phases (Liesz et al., 2013; Shi et al., 2021).

Recent studies have found that long-term accumulation of CD8+ T cells after brain injury leads to prolonged neuronal damage (Daglas et al., 2019), and delayed removal of CD8+ T cells can improve functional recovery after stroke (Selvaraj et al., 2021). However, the specific roles of these T cell subsets in post-stroke neuroinflammation remain unclear. This paper fills this research gap by exploring the potential roles of CD8+ T cell subsets in long-term neuroinflammation after stroke.

Paper Source

The authors of this paper are Long Shu, Hui Xu, Jiale Ji, Yuhan Xu, Ziyue Dong, Yuchen Wu, and Yijing Guo, from the Department of Neurology, Zhongda Hospital, Southeast University, and the Department of Neurology, Renhe Hospital Affiliated to China Three Gorges University. The paper was accepted by the journal “Neuromolecular Medicine” on April 9, 2024, and published in Volume 26, Page 17, 2024.

Research Process

This study used C57BL/6J mice as experimental subjects and established a transient middle cerebral artery occlusion (tmcao) model. The experimental process included the following steps:

1. Establishment of tmcao model:

   • Mice underwent surgery under 2.0% isoflurane anesthesia, using a silicone-coated monofilament advanced into the left internal carotid artery to the origin of the middle cerebral artery for 90 minutes of occlusion. Cerebral blood flow (CBF) was monitored throughout the surgery as a criterion for successful operation.
   • Post-surgery, mice were divided into two groups: the first group had spleen cells and immune cells from the injured side collected on day 3 post-surgery, while the second group underwent the same procedure on day 30 post-surgery.

2. Collection and processing of immune cells:

   • Mice were euthanized with CO2 and underwent cardiac perfusion to remove circulating blood cells. The spleen and injured side of the brain were cut into small pieces and digested. The digested single-cell suspension was separated using lymphocyte separation fluid and used for subsequent experiments.

3. Flow cytometry analysis:

   • Collected cells were stained for surface markers and intracellular proteins, analyzed using a BD LSR ii flow cytometer, and CD8+ T cell subsets were isolated. Primary cell activation was performed by adding PMA and ionomycin.

4. RNA extraction and real-time PCR:

   • Total RNA was extracted using an RNA extraction kit, reverse transcribed to synthesize cDNA, followed by real-time PCR to quantify transcription levels of inflammatory mediators.

5. Co-culture experiments:

   • To verify the effects of CD8+ T cell subsets on microglia. Isolated CD8+ T cell subsets were co-cultured with CD45lowCD11b+ microglia from normal mouse brains for 24 hours, followed by RNA extraction from co-cultured microglia for real-time PCR analysis.

6. Immunofluorescence imaging:

   • Immunofluorescence labeling was used to verify the expression of CD8+ T cells and GFAP (astrocyte marker) in brain tissue sections and cultured microglia.

Research Results

Long-term presence of CD8+ T cells

Immune cells were collected from the spleen and injured side of the brain on day 3 and day 30 post-surgery. Results showed that the number of CD8+ T cells in the injured side of the brain increased significantly over time and was mainly concentrated in the peri-infarct area.

Cytokine expression

Flow cytometry analysis revealed that on day 3 post-surgery, CD8+ T cells in the spleen hardly expressed IFN-γ and IL-17A, while on day 30, CD8+ T cells in the spleen mainly produced IFN-γ. CD8+ T cells in the injured side of the brain on day 3 post-surgery were also mainly IFN-γ-producing cells, while on day 30, CD8+ T cells in the injured side of the brain included IFN-γ+ / IL-17A+ double-positive cells.

Identification of TC1 and TC17 cells in the injured side of the brain

Further analysis showed that TC1 cells were already present in the injured side of the brain on day 3 post-surgery, while on day 30, IL-17A positive TC17 cells and IFN-γ and IL-17A double-positive TC17/1 cells were also detected.

Identification and enrichment of live TC1, TC17, and TC17/1 cells

The study successfully identified and isolated TC1, TC17, and TC17/1 cells in the injured side of the brain on day 30 post-surgery using known chemokine/cytokine receptor markers. TC1 cells were defined as CD8+ CCR6- CXCR3+ cells, TC17 cells as CD8+ CCR6hi CXCR3-/lo CCR4+ IL-23R+ cells, and TC17/1 cells as CD8+ CCR6int CXCR3+CCR4+ IL-23R+ cells.

TC17/1 cells induce the strongest inflammatory response in microglia

Co-culture experiments showed that all three CD8+ T cell subsets could activate microglia and induce the expression of inflammatory mediators, with TC17/1 cells having the strongest inductive effect.

Research Conclusions

This study revealed for the first time the potential roles of CD8+ T cell subsets (including TC1, TC17, and TC17/1 cells) in long-term neuroinflammation after stroke. These cell subsets, especially TC17/1 cells, may play an important role in chronic neuroinflammation after stroke by activating microglia, thus potentially becoming new targets for future stroke treatment.

Research Highlights

1. First discovery: First report on the long-term presence and role of TC1, TC17, and TC17/1 cells in the brain after stroke.
2. Cell identification: Successfully identified and enriched live TC1, TC17, and TC17/1 cells and verified their marker expression.
3. Functional verification: Verified the different roles of these cell subsets in activating microglia and inducing inflammatory responses through co-culture experiments, finding that TC17/1 cells have the strongest inductive effect.

This study deepens our understanding of the long-term neuroinflammation process after stroke and suggests new therapeutic approaches. Future research needs to further explore the origin, dynamic changes of these cell subsets, and their effects on other neural cells and functional recovery.