T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers

Abstract

Background: Trigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (T_RM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ.

Methods: Peripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis.

Results: Collagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical T_RM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher T_RM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1⁺), which occasionally showed features of proliferation (KI-67⁺) and activation (CD137⁺), but without signs of degranulation (CD107a⁺) nor damage (TUNEL⁺) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a⁺) were not detected.

Conclusions: The human TG represents an immunocompetent environment in which both CD4 and CD8 T_RM are established and retained. Based on our study insights, we advocate for T_RM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

Keywords: Human; Normal-appearing white matter; Tissue-resident memory T-cells and herpes simplex virus; Trigeminal ganglion.