Abstract
Purpose :
In this study, we used a flow cytometry-based approach to quantify and functionally evaluate the influx of immune cells in the separated epithelium versus stroma of HSV-1 infected corneas from C57BL/6J mice during the lytic and latent phase of infection.
Methods :
The corneas of eight-twelve weeks old C57BL/6J mice were mildly scratched, followed by the topical application of HSV-1 McKrae or HSV-1 KOS. Mice were euthanized at 3-, 6-, 8-, 16-, 30-, and 56-day post-infection (p.i.). Single-cell suspensions were prepared from the separated corneal epithelium (CE) and corneal stroma (CS) for flow cytometry assay. The effector functions of memory T cells were assessed by PMA/ionomycin stimulation followed by intracellular cytokine staining (ICCS).
Results :
Our results showed the biphasic influx of CD45+ leukocytes in the CE, whereas a progressive increase in CD45+ cells was seen in the stroma at 3-, 6-, 8-, and 16-day p.i. In the CE, the frequency of myeloid cells was significantly higher at 3- than 6- and 8-day p.i. At 3-day p.i., most myeloid cells were either monocyte or neutrophils, whereas at 6-day p.i., almost all myeloid cells in the epithelium were either monocytes or macrophages. Most leukocytes in the CS at 3-, 6-, and 8-day p.i. were expressing CD11b molecule, and among them, monocytes were the predominant immune cell type. Interestingly, in CE, monocytes were significantly higher at 3-day p.i. compared to 6-day p.i., whereas a reverse trend was observed in CS. At 8-day p.i., TCR-beta+ T cells were seen in both CE and CS, but the CS had more short-lived effector T cells (KLRG1+) than CE. On the contrary, CE was the preferential homing site for tissue-resident memory (TRM) CD103+TCR-beta+ T cells at 30-day p.i. Among the TRM, we observed the predominance of CD4+ T cells over CD8+ T cells in CE. Additionally, the ICCS assay showed the production of TNF-alpha, IL-2, and IFN-γ cytokines from these memory T cells documenting their functional status.
Conclusions :
Collectively, our results showed a differential influx of monocytes in the epithelium versus stroma during the lytic phase of infection, suggesting their involvement in viral clearance. Besides, the predominance of functional tissue-resident memory CD4 over CD8 T cells is seen in the epithelium during the latent phase of infection.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.