Herpes Stromal Keratitis (HSK) development erodes the establishment of Herpes Simplex Virus-1 (HSV-1) induced tissue-resident memory T (T_RM) pool in inflamed corneas.

Abstract

Purpose : This study aimed to enumerate the T_RM pool in HSV-1 infected corneas displaying either no opacity and hemangiogenesis (non-HSK cornea) or severe opacity and hemangiogenesis (HSK cornea).

Methods : The corneas of C57BL/6J mice were mildly scratched, followed by the topical application of 2X10⁶ pfu of HSV-1 KOS. Slit lamp microscopy was used to categorize HSK and non-HSK corneas. Mice were euthanized during the memory phase, 38-136 days post-infection (DPI). The corneal epithelium (CE) was separated from the underlying stroma, followed by the preparation of single-cell suspensions. Flow cytometry assays were employed to characterize and quantify the T_RM population in the CE. The effector function of T_RM was assessed by PMA/ionomycin stimulation followed by intracellular cytokine staining (ICCS). RT-qPCR quantified the latency-associated transcript (LAT) copy number in the latently infected mice’s trigeminal ganglia (TG).

Results : The data from ten experiments showed that approximately 61.7±3.61% of the infected corneas exhibited a non-HSK, and 38.2±3.61% showed an HSK phenotype. The presence of LAT mRNA in the latent TGs of infected mice with non-HSK corneas suggested the occurrence of corneal infection. At 56-DPI, the CE of non-HSK corneas displayed a significantly higher number of CD45+ leukocytes and a greater frequency and number of TCR-b+ T cells than HSK corneas. Our results showed that more than 90% of TCR-b+ T cells in non-HSK and HSK corneas expressed CD49a, a key marker of T_RM in the barrier tissue. Interestingly, non-HSK corneas exhibited a significantly increased proportion (55.5± 2.3) of CD69+ CD103+ T_RM than HSK corneas (22.9±1.2). The numbers of CD4+ and CD8+ T cells were significantly higher in non-HSK than in HSK corneas at the memory phase. Among CD8 T cells, there was a predominance of CD69+CD103+ T_RM subset, whereas most CD4 T cells exhibited CD69+CD103- phenotype. PD-1 expression was noted on both CD4 and CD8 T_RM. However, PMA/Ionomycin stimulation of CD4 and CD8 T cells from non-HSK and HSK corneas documented their ability to produce TNF-α, IL-2, and IFNγ cytokines.

Conclusions : The quantitatively depleted T_RM pool in HSK corneas can be the outcome of the loss of the T_RM supportive environment in the corneas that develop HSK. The ongoing experiments are addressing this concept.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.