Antigen-specific polyfunctional cytotoxic T cells differentiate intraocular from peripheral blood immune responses in...

Peripheral blood is frequently used to study the immune response in human uveitis because of the inaccessibility of ocular tissue samples. To determine whether peripheral blood immune cells accurately reflect the intraocular immune response, we compared the T-cell profiles and antigen-specific cytokine responses between paired vitreous and peripheral blood samples from patients with sight-threatening posterior uveitis.

We collected paired vitreous and peripheral blood mononuclear cells (PBMCs) from 24 patients with posterior uveitis. Multi-parametric flow cytometry was employed to identify surface and intracellular cytokine markers after activation with candidate antigenic peptides [Mycobacterium tuberculosis (MTb) peptides and retinal autoantigens]. Data were analysed through manual gating, unsupervised clustering and dimensionality reduction (FlowSOM, FlowJo).

The CD8⁺/CD4⁺ ratio in a representative set of seven paired samples was higher in the vitreous than in PBMCs. Vitreous CD4⁺ and CD8⁺ cells displayed greater polyfunctional potential (TNFα⁺IFNγ⁺IL-2⁺ and PMA/ionomycin activation) than PBMCs. Upon antigen-specific activation in vitro, vitreous CD8⁺ T cells (but not CD4⁺ T cells) showed a stronger polyfunctional response than PBMCs against both MTb (in TB-immunoreactive patients) and retinal autoantigens. Unsupervised clustering identified 15 distinct CD3⁺ T-cell metaclusters, each with unique profiles in the vitreous and PBMCs. Significant cluster enrichment was observed among the vitreous infiltrating cells in TB-immunoreactive cases compared to non-TB uveitis, but no such enrichment was found among PBMCs in either patient cohort.

The vitreous T-cell compartment in this group of uveitis patients was functionally dominated by antigen-responsive cytotoxic CD8⁺ T cells and was distinct from the corresponding peripheral blood compartment.