Tissue resident memory T cells inhabit the deep human conjunctiva.

Mucosal linings of the body, including the conjunctiva, are enriched in tissue-resident memory T cells (TRMs) whose defining feature is their continual tissue protection that does not rely on migration to lymphoid organs to elicit immune responses. Hitherto, conjunctival TRMs have only been identified in the superficial epithelium. This work aims to develop a more complete understanding of the conjunctival immunological capacity by investigating the presence of TRMs within the deeper, more stable layers of the healthy human conjunctiva. Using immunofluorescence microscopy and antibodies against CD3, CD4, CD69 and HLA-DR on bulbar conjunctival biopsies obtained from 7 healthy adults (age range = 32-77 years; females = 4), we identified CD69+TRM subsets in all layers of the human conjunctiva: the superficial epithelium, the basal epithelium, the adenoid, and the fibrous layers. Interestingly, the adenoid layer showed significantly higher densities of both CD4 and CD8 TRMs when compared to the fibrous layer and conjunctival epithelia. Additionally, CD4 TRMs predominated significantly over CD8 TRMs in the adenoid layer. The abundance of deep conjunctival CD69+TRMs within the healthy human may suggest the presence of defence mechanisms capable of inducing long-term immunogenic memory. Understanding this spatial distribution of conjunctival CD69+TRMs is essential to improving mucosal vaccine design.