CD8+ T cell infiltration and proliferation in the brainstem during experimental cerebral malaria

Abstract

Introduction: Cerebral malaria (CM) is a lethal neuroinflammatory disease caused by Plasmodium infection. Immune cells and brain parenchyma cells contribute to the pathogenesis of CM. However, a systematic examination of the changes that occur in the brain parenchyma region during CM at the single-cell resolution is still poorly studied.

Aims: To explore cell composition and CD8⁺ T cell infiltration, single-cell RNA sequencing (scRNA-seq) was performed on the brainstems of healthy and experimental cerebral malaria (ECM) mice. Then CD8⁺ T cell infiltration was confirmed by flow cytometry and immunofluorescence assays. Subsequently, the characteristics of the brain-infiltrated CD8⁺ T cells were analyzed. Finally, the interactions between parenchyma cells and brain-infiltrated CD8⁺ T cells were studied with an astrocytes-CD8⁺ T cell cocultured model.

Results: The brainstem is the most severely damaged site during ECM. ScRNA-seq revealed a large number of CD8⁺ T cells infiltrating into the brainstem in ECM mice. Brain-infiltrated CD8⁺ T cells were highly activated according to scRNA-seq, immunofluorescence, and flow cytometry assays. Further analysis found a subset of ki-67⁺ CD8⁺ T cells that have a higher transcriptional level of genes related to T cell function, activation, and proliferation, suggesting that they were exposed to specific antigens presented by brain parenchyma cells. Brain-infiltrated CD8⁺ T cells were the only prominent source of IFN-γ in this single-cell analysis. Astrocytes, which have a high interferon response, act as cross-presenting cells to recruit and re-activate brain-infiltrated CD8⁺ T cells. We also found that brain-infiltrated CD8⁺ T cells were highly expressed immune checkpoint molecule PD-1, while parenchyma cells showed up-regulation of PD-L1 after infection.

Conclusions: These findings reveal a novel interaction between brain-infiltrated CD8⁺ T cells and parenchyma cells in the ECM brainstem, suggesting that the PD-1/PD-L1 signal pathway is a promising adjunctive therapeutic strategy for ECM targeting over-activated CD8⁺ T cells.

Keywords: PD-1/PD-L1 pathway; astrocytes; brain-infiltrated CD8+ T cell; brainstem; experimental cerebral malaria; single-cell RNA sequencing.