Loss of Ocular Mucosal Tolerance with Aging is Accompanied by Changes in T Regulatory Transcriptome

DE PAIVA, CINTIA S.; TRUJILLO-VARGAS, CLAUDIA M.; HERNANDEZ, HUMBERTO; DE SOUZA, RODRIGO G.; YU, ZHIYUAN; GALLETTI JEREMÍAS GASTÓN

Congreso; ARVO 2020 ANNUAL MEETING; 2020

ARVO

Purpose : The purpose was to investigate the generation of antigen-specific regulatory cells (Tregs) with aging.Methods : Young (8-week-old, 8W) and aged (24-months old, 24M) female C57BL/6J mice were used. Mice received Ovalbumin (OVA) for five consecutive days. On the 7th day, CD4+ T cells were isolated from cervical lymph nodes and spleens. The frequency of CD45+CD4+CD25+CD304+ cells (inducible Tregs) was investigated in draining nodes by flow cytometry. CD4+CD25+GITR+ and CD4+CD25-GITR- cells were sorted and used as surrogates for Tregs and T responders (Tresps). Tregs and Tresps were plated at different cell concentrations with Violet-tracer?-labelled CD4+ OT-II cells, OVA, and CD3-depleted splenocytes. Proliferation was investigated by flow cytometry. A separate group of young and aged mice was used in sorting experiments to obtain CD4+CD25+GITR+ and CD4+CD25-GITR- cells from spleens and nodes. Cells were lyzed, and mRNA was later used for transcriptome analysis using Nanostring? technology.Results : After OVA eyedrops, aged mice had increased frequency of CD4+CD25+ T cells and lower frequency of CD4+CD25+CD304- cells in draining nodes than young mice. In antigen-specific assays using OVA as a surrogate antigen, OVA-24M mice had decreased suppressive capacity OVA-8W Tregs. Nanostring analysis showed profound changes in the transcriptome of both Tresps and Tregs with aging. There was a decrease in Sell, Lef1, CCR7 (naïve markers), an increase in CD44, Itgb1 (T effector memory), granzymes A and B, perforin, IL-21 and Runx3 (cytotoxic markers) and Tnfsf8, Prdm1, Pdcd1, Nfatc1 and Eomes (exhausted markers) in 24M Tresps. Aged Tregs expressed higher levels of IFN-gamma, IL-17, but still expressed significantly higher levels of IL-10, CTLA-4 and ICOS mRNA transcripts than young Tregs. Furthermore, aged Tregs acquired, CCR5, CXCR3 and Tbet expression; markers associated with a Th1 profile and showed an activated phenotype, as there was a significant increase in CD81, CD74, Tnfrsf4, Ikzf2, Tnfrsf9, relb, and NFkb2 genes.Conclusions : Aged mice failed to generate antigen-specific Tregs after topical administration, suggesting loss of ocular mucosal tolerance. Aging was accompanied by profound changes in the transcriptome of aged effectors and aged Tregs, suggesting a defect in both cell types. These findings indicate that age-related changes might participate in the pathogenesis of dry eye.