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 (T_RMs) whose defining feature is their continual tissue protection that does not rely on migration to lymphoid organs to elicit immune responses. Hitherto, conjunctival T_RMs 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 T_RMs 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⁺T_RM 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 T_RMs when compared to the fibrous layer and conjunctival epithelia. Additionally, CD4 T_RMs predominated significantly over CD8 T_RMs in the adenoid layer. The abundance of deep conjunctival CD69⁺T_RMs 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⁺T_RMs is essential to improving mucosal vaccine design.