Corneas Exposed to Desiccating Stress Are Immunogenic and Induce T Cell Proliferation in Mice With Experimental Lacrimal Keratoconjunctivitis (LKC)

Abstract

Purpose: : Environmental desiccating stress (DS) has been found to initiate CD4⁺ T cell-mediated lacrimal keratoconjunctivitis (LKC). Infiltrating T cells were restricted to the Lacrimal Functional Unit (LFU, cornea, conjunctiva and lacrimal gland) after adoptively transferred from mice exposed to DS to T cell deficient nude mice. We hypothesize that this ocular surface (OS) inflammation is engendered by DS-induced epithelial immunogenicity in the components of LFU.

Methods: : From naive or DS mice (BALB/c), CD4⁺ T cells were purified from spleen/cervical lymph nodes and co-cultured with full-thickness cornea wedges for 4 days. T cell proliferation was quantified by WST assay. Co-culture was carried out in the presence or absence of anti-mouse I-A/I-E mAb (1 µg/10⁶ CD4⁺ T cells) to determine if T cell response to DS cornea stimulation was due to MHC class II-bearing antigen presenting cells present in the DS cornea. This was further studied in vivo by i.p. injection to mice with the same anti-MHC II mAb on days-3, +1 and +3 of DS induction (100 µg Ab/mouse/dose). Corneas were replaced by dendritic cells (DC, differentiated from mouse bone marrow and pulsed with total corneal proteins) in the CD4⁺ T cells co-culture (DC:CD4⁺=1:10) to investigate the potential antigenic role of ocular surface in LKC. Cytokines in the culture supernatant were determined by Luminex.

Results: : CD4⁺ T cell proliferation was induced by corneas of DS mice in comparison to corneas of naive mice (52% increase, p=2.18E-05). DS cornea-induced CD4⁺ T cell proliferation was inhibited when co-culture was carried out in the presence of anti MHC class II mAbs (80% decrease, p=0.0044) as compared to the isotype control. In vivo administration of anti-MHC class II Ab in naïve BALB/c mice prior to expose to DS also inhibited CD4⁺ T cell proliferation (41% decrease, p=0.012). Cytokines (IL-6, TNF-a, and GM-CSF) were significantly increased (p<0.02) in the supernatants from CD4⁺ T cells and DS cornea co-culture, but decreased in cultures treated with anti-MHC II mAbs. When DS cornea was replaced by DCs-paused with DS corneal proteins, a similar proliferative response from CD4⁺ T cells was detected.

Conclusions: : The enhanced lymphoproliferation induced by DS corneas suggests an immunogenic basis for the desiccated ocular surface in promoting T cell activation in the pathogenesis of LKC.

Keywords: cornea: tears/tear film/dry eye • inflammation • antigen presentation/processing