Terminally differentiated cytotoxic CD4+ T cells were clonally expanded in the brain lesion of radiation-induced brain injury

Abstract

Background: Accumulating evidence supports the involvement of adaptive immunity in the development of radiation-induced brain injury (RIBI). Our previous work has emphasized the cytotoxic function of CD8⁺ T cells in RIBI. In this study, we aimed to investigate the presence and potential roles of cytotoxic CD4⁺ T cells (CD4⁺ CTLs) in RIBI to gain a more comprehensive understanding of adaptive immunity in this context.

Main text: Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed 3934 CD4⁺ T cells from the brain lesions of four RIBI patients and identified six subclusters within this population. A notable subset, the cytotoxic CD4⁺ T cells (CD4⁺ CTLs), was marked with high expression of cytotoxicity-related genes (NKG7, GZMH, GNLY, FGFBP2, and GZMB) and several chemokine and chemokine receptors (CCL5, CX3CR1, and CCL4L2). Through in-depth pseudotime analysis, which simulates the development of CD4⁺ T cells, we observed that the CD4⁺ CTLs exhibited signatures of terminal differentiation. Their functions were enriched in protein serine/threonine kinase activity, GTPase regulator activity, phosphoprotein phosphatase activity, and cysteine-type endopeptidase activity involved in the apoptotic signaling pathway. Correspondingly, mice subjected to gamma knife irradiation on the brain showed a time-dependent infiltration of CD4⁺ T cells, an increase of MHCII⁺ cells, and the existence of CD4⁺ CTLs in lesions, along with an elevation of apoptotic-related proteins. Finally, and most crucially, single-cell T-cell receptor sequencing (scTCR-seq) analysis at the patient level determined a large clonal expansion of CD4⁺ CTLs in lesion tissues of RIBI. Transcriptional factor-encoding genes TBX21, RORB, and EOMES showed positive correlations with the cytotoxic functions of CD4⁺ T cells, suggesting their potential to distinguish RIBI-related CD4⁺ CTLs from other subsets.

Conclusion: The present study enriches the understanding of the transcriptional landscape of adaptive immune cells in RIBI patients. It provides the first description of a clonally expanded CD4⁺ CTL subset in RIBI lesions, which may illuminate new mechanisms in the development of RIBI and offer potential biomarkers or therapeutic targets for the disease.

Keywords: CD4+ CTL; TCR sequencing; adaptive immunity; clonal expansion; radiation-induced brain injury.