A pilot study to characterise the immune response in human Herpes Keratitis

Abstract

Purpose : Herpes Keratitis (HK) is a corneal infection causing the highest rate of infectious blindness in developed countries. The immune response to HK in human tissue has not been widely studied to date. This study aims to characterise the different subsets and locations of immune cells in human corneal tissue and compare them between herpetic and cadaver corneas.

Methods : Six corneas from cadavers were collected from NSW Tissue Banks and 1 HK sample was collected from Sydney Eye Hospital. ¾ segments of each cornea was incubated with Collagenase Type II for 4 hours to extract cells from the epithelium and stroma in a single suspension before staining with monoclonal anti-human antibodies and analysing them by high parameter flow cytometry. The 23 markers include CD14, CD11c, CD4, CD8 and CD56. Results are presented as mean (± SEM).
The remaining ¼ segments from each cornea were fixed in 4% paraformaldehyde for 24 hours prior to staining with fluorescently labelled DAPI, langerin, CD3 and CD56. Sections were imaged to assess the locations and morphologies of Langerhans cells, T cells and natural killer cells.

Results : Epithelia of the cadaver samples (time post-enucleation range 0-1 days) showed an increased presence of CD14⁺ macrophages (10.46 ± 2.23) and conventional type 2 dendritic cells (13.94 ± 7.43) compared to the HK corneal sample (1.56; 0.28 respectively). However, CD4 cells, CD8 T cells and natural killer cells were shown at higher proportions in the epithelium of the HK sample (21.4; 31.5; 16.1 respectively) compared to the cadaver control groups (19.74 ± 7.86; 26.97 ± 5.11; 2.97 ± 1.14). Similar trends were seen in the stroma however, there was no difference between natural killer cell populations.
Immunofluorescence microscopy of the HK cornea showed that there was an accumulation of T cells in the central region of the stroma (59) whereas human cadaver samples showed minimal T cells in this corresponding region (3.33 ± 1.67). Human cadaver samples showed Langerhans’ cells, T cells and natural killer cells residing in the limbal epithelium and stroma as previously shown.

Conclusions : Human HK tissue exhibited a decrease in myeloid cells and increase in lymphocytes compared to cadaver samples, which were mainly located in the central stroma. Further investigation is required to better characterise the role of these cell types and the pathway of myeloid cells in human HK.

This is a 2021 ARVO Annual Meeting abstract.